Stromal Complement Receptor CD21/35 Facilitates Lymphoid Prion Colonization and Pathogenesis

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1 This information is current as of December 30, References Subscription Permissions Alerts Stromal Complement Receptor CD21/35 Facilitates Lymphoid Prion Colonization and Pathogenesis Mark D. Zabel, Mathias Heikenwalder, Marco Prinz, Isabelle Arrighi, Petra Schwarz, Jan Kranich, Adriana von Teichman, Karen M. Haas, Nicolas Zeller, Thomas F. Tedder, John H. Weis and Adriano Aguzzi J Immunol 2007; 179: ; ; doi: /jimmunol This article cites 74 articles, 25 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on December 30, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2007 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology Stromal Complement Receptor CD21/35 Facilitates Lymphoid Prion Colonization and Pathogenesis 1 Mark D. Zabel, 2 * Mathias Heikenwalder,* Marco Prinz, Isabelle Arrighi,* Petra Schwarz,* Jan Kranich,* Adriana von Teichman,* Karen M. Haas, Nicolas Zeller,* Thomas F. Tedder, John H. Weis, and Adriano Aguzzi 2 * We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35 / mice experienced lower attack rates and delayed disease over both wild-type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35 / spleens were devoid of infectivity. Reciprocal adoptive bone marrow transfers between WT and CD21/35 / mice revealed that protection from prion infection resulted from ablation of stromal, but not hemopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrP C and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Because both PrP C and CD21/35 are highly expressed on follicular dendritic cells, CD21/35 appears to be involved in targeting prions to follicular dendritic cells and expediting neuroinvasion following peripheral exposure to prions. The Journal of Immunology, 2007, 179: Transmissible spongiform encephalopathies (TSEs) 3 are fatal neurodegenerative diseases for which no early diagnosis or treatment other than palliation exists. Accumulation of PrP Sc, a proteinase K (PK) resistant form of the normal cellular prion protein, PrP C, is common to most instances of TSEs. Interspecies transmission was seen in TSEs both in the field and in the laboratory, and transmission from bovine spongiform encephalopathy-infected cattle to humans has almost certainly occurred (1 5). The infectious agent, termed prion, has been detected in nervous, lymphoid, and muscle tissues (6 12), as well as blood, urine, and saliva (13 20). Chronic wasting disease in cervids and scrapie in sheep appear to be unique among TSEs because of their high transmission efficiency and prevalence (21 24). *Institute for Neuropathology, University Hospital of Zürich, Zürich, Switzerland; Institute of Neuropathology, Georg-August University, Göttingen, Germany; Department of Immunology, Duke University Medical Center, Durham, NC 27710; and Department of Pathology, University of Utah, Salt Lake City, UT Received for publication June 15, Accepted for publication August 16, 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 M.D.Z. was supported by the Human Frontiers in Science Foundation Long-Term Fellowship and the Volkswagen Foundation. M.H. was supported by grants of the Bonizzi-Theler Foundation, the Schweizer MS Foundation, and the Prof. Max-Cloëtta Foundation. A.A. was supported by the Volkswagen Foundation, the Swiss National Foundation, and the Ernst-Jung Foundation. 2 Address correspondence and reprint requests to Dr. Mark D. Zabel at the current address: Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 1619 Campus Delivery, Fort Collins, CO ; address: mark.zabel@colostate.edu or Dr. Adriano Aguzzi, Institute of Neuropathology, Department of Pathology, Universitätspital Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland; address: adriano.aguzzi@usz.ch 3 Abbreviations used in this paper: TSE, transmissible spongiform encephalopathy; PK, proteinase K; FDC, follicular dendritic cell; BM, bone marrow; dpi, days postinoculation; SCEPA, scrapie cell assay in endpoint format; WT, wild type; IHC, immunohistochemistry; MBA, mouse bioassay; i.c., intracerebral; NaPTA, sodium phosphotungstic acid; IF, immunofluorescence stain; PrP, prion protein; PrP C, cellular prion protein; PrP Sc, scrapie-associated, misfolded prion protein. Copyright 2007 by The American Association of Immunologists, Inc /07/$2.00 Extracerebral prion accumulation and replication precedes neuroinvasion in many cases of TSEs. The immune system plays an important role in PrP Sc neuroinvasion from peripheral sites in murine models of scrapie (25 27). Prion accumulation and replication occur in lymphoid follicles or inflammatory foci containing follicular dendritic cells (FDCs) that express PrP C (28 32). FDCs are cells of stromal origin that trap immune complexes on their elaborate projections and present them to B cells, with which they closely associate (33 37). They may retain Ag on their cell surfaces for prolonged periods, maximizing presentation to B cells. FDCs accumulate PrP-immunoreactive material, and depletion of FDCs suppresses lymphoid prion titers. These observations suggest that FDCs replicate prions (30), although this has not been formally proven. Splenic PrP Sc accumulation requires B cells that, despite replicating little or no PrP Sc themselves, are required for neuroinvasion (38). Because most B cells express little or no PrP C (39), this requirement presumably relates to B cells supplying FDCs with lymphotoxins necessary for their maturation and maintenance (40 45). The mechanism by which FDCs trap and propagate prions is poorly understood, and it is unknown whether molecules other than PrP C are involved. Both B cells and FDCs express and interact with components of the complement system shown to be important for prion neuroinvasion and pathogenesis. The serum complement protein C1q binding immune complexes triggers the classical complement pathway. Mice deficient in C1q exhibit dramatically delayed PrP Sc accumulation and onset of terminal disease when inoculated with scrapie prions (46, 47). C1q binds PrP C in a conformation- and density-dependent manner and PrP Sc activates the classical complement pathway (48, 49). Cleavage products of the soluble complement proteins C3 and C4 covalently attach to microbial surfaces and immune complexes, which are then presented as Ags on the surface of B cells and FDCs via the complement receptors CD21/35 (50 52). Pharmacological or genetic ablation of C3 and full-length membrane-bound receptors CD21/35 delays prion pathogenesis (46, 47). These data suggest that complement may mediate FDC trapping of prions. In this study, we show that complete elimination of the complement proteins that trap Ag on FDCs significantly delays splenic prion

3 The Journal of Immunology 6145 accumulation and terminal prion disease in mice inoculated i.p. with the Rocky Mountain laboratory (RML) strain of prions. Ablation of complement receptors CD21/35 affected prion trapping and disease more profoundly than ablating their ligand sources, C3 and C4, suggesting a role for CD21/35 in peripheral prion pathogenesis independent of their endogenous ligands. To assess the relative importance of CD21/35 on hemopoietic and stromal cell types, we performed reciprocal reconstitution experiments by bone marrow (BM) transplantation. CD21/35 expression exclusively on FDCs in white pulp follicles resulted in prion titers, PrP Sc retention, and disease kinetics and severity similar to those of wild-type (WT) mice. CD21/35 expression on hemopoietic cells also significantly affected these parameters, but far less than FDC expression of CD21/35. Therefore, complementmediated Ag trapping on FDCs is an important mechanism for lymphoid prion accumulation. Materials and Methods Mice C57BL/6 129sv (B6 129 SF2/J), C3 / and C4 / (C 3tm1Crr/J and C4 tm1crr, C57BL/6 strain) mice were purchased from The Jackson Laboratory. C3 / and C4 / mice were crossed to produce C3/C4 / mice. TgA20, which overexpress mouse PrP, CD21/35 / and Prnp o/o mice have been previously described (53 55). CD21/35 / and Prnp o/o mice were crossed to produce Prnp o/o CD21/35 / mice. Breeding and experiments were performed in compliance with the animal experimentation guidelines of the Kanton of Zürich. Prion inoculations Transgenic and control (C57BL/6 129sv) animals were infected i.p. with 100 l of brain homogenate diluted in 320 mm sucrose, containing 3 or 6 log LD 50 U of the Rocky Mountain laboratory (RML) scrapie strain passage 5 (RML 5.0), the titer of which was previously assessed by intracerebral (i.c.) inoculation into TgA20 mice and found to be 8.9 log LD 50 /g of brain tissue. Mice were monitored every other day, and scrapie were diagnosed according to clinical criteria including ataxia, kyphosis, tail rigidity, and hind leg paresis. Mice were sacrificed at the onset of terminal disease. Infectivity mouse bioassay (MBA) Assays were performed on 1% spleen homogenates. Spleen tissues were homogenized in sterile 320 mm sucrose (1/10) with a RiboLyser (Hybaid), centrifuged for 5 min at 500 g. Cleared supernatants were diluted 1/10 in sterile 5% BSA in PBS and 30 l was injected i.c. into each of four TgA20 mice per homogenate (55). Titers were determined using the relationship: y x, where y is log LD 50 and x is the incubation time in days to terminal disease (56). Histological and immunohistochemical analyses using H&E, glial fibrillary acidic protein, and SAF84 staining revealed spongiosis, gliosis, and PrP deposition in all scrapie symptomatic mice and none of these histopathological features in asymptomatic mice sacrificed days postinoculation (dpi). Scrapie cell assay in endpoint format (SCEPA) Replicate aliquots of highly prion-susceptible neuroblastoma cells (subclone N2aPK1; Ref. 57) were placed into 12 wells of a 96-well plate and exposed to prion samples for 3 days, split 1:3 three times every 2 days, and 1:10 three times every 3 days. After reaching confluence, cells from each well were filtered onto the membrane of an ELISPOT plate, treated with PK, denatured, and individual infected (PrP Sc -positive) cells were detected immunochemically using Ab ICSM-18 to PrP. Wells were scored positive if the spot number exceeded mean background values plus five times the SD. From the proportion of negative to total wells, the number of infectious tissue culture units per aliquot was calculated using the Poisson equation. The potency of the SCEPA is based on the finding that the proportion of infected cells, and with it the signal-to-background ratio, increases on average 25% per day during culturing (57). Histology and immunohistochemistry (IHC) A total of 2 m of paraffin or 5 10 m of frozen sections from brain and spleen were stained with H&E. Infected, formalin-fixed brain samples were treated with concentrated formic acid for 60 min to inactivate prion infectivity. Astrocytes were stained with rabbit anti-glial fibrillary acidic protein mab (DakoCytomation; 1/300) and visualized with biotinylated swine serum against rabbit IgG (DakoCytomation; 1/250), avidin-peroxidase (DakoCytomation), and diaminobenzidine (Sigma-Aldrich). PrP was visualized with anti-prp SAF-84 mab (A03208, 1/200; SPI Bio). FDCs were stained with anti-fdc-m1 mab (clone 4C11, 1/50; BD Biosciences) and B cells and FDCs, with rat anti-cd21/35 mab (7G6, 1/100; BD Pharmingen) on frozen acetone-fixed sections of spleens and visualized by incubation with goat Ab against rat IgG (Milan) and alkaline phosphataseconjugated donkey Abs against goat IgG with fast red. Pairs of consecutive sections were used to quantify CD21/35 and FDC-M1 expression on individual splenic follicles from three distinct areas in spleens from two mice per group. Quantification of CD21/35 and FDC-M1 IHC signal intensities was performed using the CMYK color model (58) in Adobe Photoshop. Two-color immunofluorescence was performed with rabbit anti-mouse PrP mab XN (1/1000) and FITC-conjugated rat anti-mouse CD21/35 mab 7G6 (1/100) on frozen acetone-fixed spleen sections. Alexa 546-conjugated goat IgG against rabbit IgG (Molecular Probes) was used to visualize PrP. For controls, preimmune sera were used or primary Abs were omitted. Histoblot analysis was performed as previously described (59). Briefly, frozen spleen sections were transferred to nitrocellulose membranes soaked in lysis buffer using 60 s of constant pressure. Membranes were dried overnight, soaked in TBST for 1 h, then incubated in digestion buffer containing g/ml proteinase K for 4 h. Membranes were rinsed three times for 10 min each in TBST, blocked for 1 h in 5% milk, incubated overnight with anti-prp Ab 6H4 (1/2000; Prionics), washed again with TBST, and incubated 1 h with alkaline phosphatase-goat anti-mouse IgG1 (DO486, 1/1000; DakoCytomation) and washed a final time. PrP signals were developed with 5-bromo-4-chloro-3- indolyl phosphate/nitro-blue tetrazolium chloride for 1 h. BM reconstitution and FACS analysis BM was taken from tibiae and femurs of respective donor groups and 10 7 cells were injected into tail veins of lethally irradiated (1100 radians for 10 min) recipient mice as previously described (60). Six to 8 weeks after reconstitution, peripheral blood cells from mice of each group were stained at 4 C with 1/100 dilutions of FITC-labeled rat anti-mouse CD21/35 Ab 7G6, PE-labeled rat anti-mouse B220 mab (BD Pharmingen) or Cy5-labeled mouse anti-mouse PrP mab (POM-1) in FACS buffer (0.1% BSA, 10 mm EDTA in PBS). RBC were lysed using FACSLyse solution and the remaining cells analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences). Live lymphocytes were gated based on forward scatter and side scatter properties and analyzed for CD21/35, B220, and/or PrP expression using FlowJo software. Sodium phosphotungstic acid (NaPTA) precipitation of PrP Sc Ten percent spleen homogenates were prepared in PBS as described above. Gross cellular debris was removed by centrifugation at 80 g and 500 l of supernatant mixed 1:1 with 4% sarkosyl in PBS. Samples were incubated for 15 min at 37 C with constant agitation, then incubated with 50 U/ml benzonase and mm MgCl 2 for 30 min at 37 C with constant agitation. Prewarmed NaPTA stock solution (ph 7.4) was added to a final concentration of 0.3% and the sample was incubated at 37 C for 30 min with constant agitation and centrifuged at 37 C for 30 min at maximum speed in an Eppendorf microcentrifuge. The pellet was resuspended in 30 l of 0.1% sarkosyl in PBS and digested with 20 g/ml PK for 30 min at 37 C. Immunoblot analysis Tissue homogenates were NaPTA precipitated or adjusted to 5 mg/ml protein and 50 g treated or not with 20 g/ml PK for 30 min at 37 C. Samples were heated at 95 C for 5 min in SDS-PAGE loading buffer and pipetted into wells of 12% or 4 12% gradient Novex SDS polyacrylamide gels (Invitrogen Life Technologies) and electrophoresed. Proteins were transferred to nitrocellulose membranes (Schleicher-Schuell) by wet blotting, blocked with TBST containing 5% Topblock (Juro) incubated with anti-mouse PrP mab POM-1 (1/2000) overnight at 4 C. Membranes were washed and incubated with HRP-conjugated rabbit anti-mouse IgG1 for 1 h at room temperature. Bands were detected by chemiluminescence (Pierce) and visualized by the VersaDoc imaging system (Bio-Rad). Bands were quantified using QuantityOne software (Bio-Rad). Statistical analyses One-way ANOVA and Student s t test were performed where appropriate using the software packages GraphPad Prism and Microsoft Excel. Results Absence of CD21/35 or its ligands delays prion disease Mice deficient for both C3 and C4 were obtained by serial crosses of C3 / and C4 / mice. The resulting C3/4 / offspring were

4 6146 CD12/35 EXPEDITES PRION NEUROINVASION Table I. Host Genotype a Prion disease progression in complement-deficient mice 6 Log LD 50 3 Log LD 50 Incidence b dpi c SD Incidence dpi SD C3 / 8/ / d C3/4 / 6/ d,e 6/ d CD21/35 / 10/ d,e 15/ e g WT 8/ / a Mice were inoculated i.p. with RML 5 prions. b Incidence Number of terminally sick mice/number of animals inoculated. c dpi, Days postinoculation to terminal disease. d Value of p 0.05 compared to WT. e Value of p 0.05 compared to C3 /. f Value of p 0.01 compared to WT. g Value of p 0.05 compared to C3/4 /. FIGURE 1. Delayed neuropathology and PrP Sc deposition in brains of CD21/35 / mice. IHC of hippocampal sections from asymptomatic CD21/ 35 / mice 242 dpi shows little or no vacuolation, astrogliosis, or PrP Sc accumulation (A C). Sections from terminally sick WT (244 dpi, D F) and CD21/ 35 / (475 dpi, G I) mice reveal similar microvacuolation (D and G) and astrogliosis (E and H), but greater PrP Sc accumulation in WT (F) than CD21/35 / (I) brain. Scale bars, 100 m. J, Western blot (WB) analysis of PrP Sc content from 20 g of brain homogenate from asymptomatic CD21/ 35 / mice and terminally sick WT and CD21/ 35 / mice. K, Quantification of band intensities from the WB in (J) confirms that brains from terminally sick WT mice contain significantly more PrP Sc than brains from asymptomatic or terminally sick CD21/35 / mice (, p 0.05). inoculated with saturating (6 log LD 50 ) or limiting (3 log LD 50 ) doses of RML 5 prions i.p. Upon high-dose prion challenge (Table I), C3 / mice contracted terminal prion disease with an incubation time similar to WT controls ( and days, respectively, n 8/group). In contrast, C3/4 / mice contracted prion disease 43 days later than WT controls ( days, n 6). Upon limiting-dose inoculation, C3 / (276 19, n 5) and C3/4 / (290 1, n 6) mice contracted disease 38 and 52 days later, respectively, than WT controls (238 8, n 15). Taken together, these data indicate an involvement of both C3 and C4 in extraneural prion pathogenesis and neuroinvasion. We next analyzed the contribution of the CD21/35 receptors. In a previous study, we had used mice that had been originally reported to lack CD21/35 expression (61). However, these mice express a surface-localized, biologically active hypomorphic isoform of CD21/35 (CD21/35 hypo ) at 40% of the level of full-length CD21/35 in WT mice (62). We reported previously that CD21/ 35 hypo mice exhibited minimally delayed incubation times after i.p. prion inoculation (46 days) with limiting doses of RML 5 prions (3 log LD 50 ) and no delay at saturating doses (46). Because the above studies used mice that express functional CD21/35 molecules, they may underestimate the contributions of CD21/35 to prion pathogenesis. Therefore, we now used mice completely deficient in CD21/35 expression (54). When inoculated i.p. with limiting doses of prions, 25% of CD21/35 / mice did not contract disease (Table I, n 20). Those mice that developed scrapie reached the terminal stage of disease ( days, n 15) 112 days later than congenic WT controls (238 8, n 8). CD21/35 / contracted disease 74 days later than C3 / (276 19, n 5) and 60 days later than C3/4 / (290 1, n 6) mice, all of which died from both groups. When inoculated with saturating doses of prions, CD21/35 / mice contracted disease (255 26, n 10) 49 days later than WT (206 8, n 8). We examined terminally sick WT and CD21/35 / mice for characteristic signs of prion neuropathology. Little or no vacuolation, astrogliosis or PrP Sc deposition was evident in sections of hippocampus from asymptomatic CD21/35 / sacrificed 242 days after inoculation (Fig. 1, A C). In contrast, microvacuolation and astrogliosis were evident in sections from terminally sick WT (Fig. 1, D and E) and CD21/35 / mice (Fig. 1, G and H). Although the pattern of PrP Sc deposition was similar, brains of WT mice accumulated more PrP Sc (Fig. 1F) than CD21/35 / brains (Fig. 1I).

5 The Journal of Immunology 6147 FIGURE 2. Transmission bioassays reveal no infectious prion titers in spleens from CD21/35 / mice at 15, 30, and 45 dpi with 3 log LD 50 U of RML 5.0 administered i.p. A, Mouse infectivity bioassays (MBA). Each symbol represents results from four TgA20 indicator mice inoculated i.c. with 30 l of spleen homogenate from WT (circles) or CD21/35 / (triangles) mice. SDs were within 0.75 log units. Data points below the dotted line represent attack rates 100%. B, Scrapie cell end point assays (SCEPA) confirm MBA. Highly scrapie-susceptible PK1 cells were incubated with 30 l of spleen homogenate from two WT (circles) or CD21 / mice (triangles) at the indicated times after i.p. inoculation (dpi). The area between the dashed horizontal lines indicates the dynamic range of the SCEPA. Data points outside these lines are off-scale. FIGURE 3. PrP Sc accumulation correlates with CD21/35 expression at 47 dpi. A, Consecutive sections display splenic follicle (H&E) that contain PrP Sc deposits (histoblots) and express both CD21/35 (green) and PrP C (red) in WT mice (IF). B, NoPrP Sc deposition was detected in splenic follicles expressing PrP C in CD21/35 / mice. Higher magnifications of the boxed areas are shown below each figure. Scale bars, 200 m. 15, 30, and 45 days earlier with 3 log LD 50 of RML 5.0 prions. Both assays detected prion infectivity in WT spleens at all three time points, but no infectivity at the detection limits for the MBA (1.5 log LD 50 ) and SCEPA (2.4 log LD 50 ) in CD21/35 / spleens. These data indicate severe impairment of prion accumulation in CD21/35 / mice soon after infection. PrP Sc colocalized with CD21/35 early after prion infection Because accumulation of prion infectivity correlated with CD21/35 expression in spleens of mice inoculated i.p. with prions, we investigated whether early accumulation of PK-resistant PrP C (PrP Sc ) correlated with CD21/35 expression in splenic follicles. We analyzed WT and CD21/35 / spleens for PrP Sc accumulation by histoblot (59). We detected PrP Sc in WT (Fig. 3A), but not We quantified the amount of PrP Sc in 20 g of brain homogenate from each group by Western blot (Fig. 1J). We detected all three PrP glycoforms, with the expected protease resistance and shift in m.w. of PrP Sc after PK digestion. Brain homogenates from terminally sick WT mice contained 15-fold more PrP Sc than homogenates from asymptomatic CD21/35 / mice sacrificed at similar time points, and 4-fold more PrP Sc than brains from terminally sick CD21/35 / mice (Fig. 1K, p 0.05). Absence of CD21/35 impaired early splenic prion accumulation The reduced attack rate or delay in terminal disease of CD21/ 35 / mice after i.p. prion challenge could stem from inefficient prion accumulation and replication in the periphery early after infection. We therefore examined prion loads in spleens of WT and CD21/35 / mice shortly after peripheral prion inoculation. We used the MBA (Fig. 2A) and scrapie cell endpoint assay (SCEPA; Fig. 2B) to determine prion titers of spleens from mice inoculated FIGURE 4. FACS analysis of PBLs from WT, CD21/35 /, and BM chimeric mice. PBLs were stained with Abs recognizing the B cell marker B220 and CD21/35, confirming presence or absence of CD21/35 on B cells. Numbers indicate percentage of double-positive cells.

6 6148 CD12/35 EXPEDITES PRION NEUROINVASION Table II. Prion disease progression in mice differentially expressing CD21/35 6 Log LD 50 3 Log LD 50 Donor BM a Host Genotype b CD21 Expression c Incidence dpi SD Incidence dpi SD CD21/35 / WT FDCs 4/ / d WT CD21/35 / B cells 8/ d 8/ e,f CD21/35 / CD21/35 / none 2/2 256, 280 2/5 312, 367 e,f WT WT B cells and FDCs 2/2 189, 195 7/ a BM was isolated from mice of the indicated genotype. b Hemopoietic systems of lethally irradiated mice of the indicated genotypes were reconstituted with donor BM. c CD21/35 expression was restricted to the indicated cell types for each reconstitution group. d Value of p 0.05 compared to WT3WT. e Value of p 0.01 compared to WT3WT. f Value of p 0.01 compared to CD21/35 / 3WT. CD21/35 / (Fig. 3B), spleens at 47 dpi. Accumulation of PrP Sc correlated with CD21/35 and PrP C expression in WT spleens (Fig. 3A, immunofluorescence stain (IF)). Loss of CD21/35 expression on FDCs delayed prion disease progression To define the cellular compartment in which CD21/35 is important for prion replication, we prepared reciprocal BM chimeric mice with CD21/35 expression restricted to hemopoietic (WT3 CD21/ 35 / mice) or stromal (CD21/35 / 3WT mice) compartments (Fig. 4). For control, WT mice were reconstituted with WT BM (WT3WT mice) and CD21/35 / mice were reconstituted with CD21/35 / BM (CD21/35 / 3CD21/35 / mice). We confirmed highly efficient BM engraftment by FACS analysis of PBLs from unmanipulated (WT and CD21/35 / ) and reconstituted mice (Fig. 4). BM from WT mice reconstituted 97% of CD21/35- expressing B cells in irradiated WT (WT3WT, 63%) and CD21/ 35 / (WT3CD21/35 /, 63%) mice compared with WT mice (65%). CD2135 / BM reconstitution eliminated 94 97% of CD21/35-expressing B cells in irradiated WT (CD21/35 / 3 WT, 5%) mice compared with CD21/35 / (1%) or CD21/ 35 / 3 CD21/35 / mice (3%), respectively. After high- or low-dose prion challenge, control chimeric WT3WT and CD21/35 / 3CD21/35 / mice displayed differences in incubation times similar to those of unmanipulated WT and CD21/35 / mice (Table II). At high-dose prion challenge, CD21/35 / 3CD21/35 / mice contracted terminal disease days later (256 and 280, n 2) than WT3WT mice (189 and 195, n 2). We observed incomplete attack rates (two of five) and a 106- to 161-day delay of terminal disease at low-dose challenge of CD21/35 / 3CD21/35 / mice (312 and 367, n 2) compared with WT3WT mice (206 12, n 7). At high-dose challenge, WT3CD21/35 / mice contracted disease days later (267 18, n 8) than WT3WT mice. At low-dose challenge, WT3CD21/35 / mice contracted disease 128 days later (334 42, n 8) than WT3WT mice and 80 days later than CD21/35 / 3WT mice (254 22, n 8). These data confirm the importance of CD21/35 expression on FDCs for facilitating terminal prion disease. Although the delay observed for these mice (334 days), which lack CD21/35 expression on FDCs, is very similar to that observed for control CD21/35 / 3CD21/35 / mice (312 and 367 days), we observed no decreased incidence (eight of eight) as we did for the controls (two of five). In addition, CD21/ 35 / 3WT mice, which lack CD21/35 expression on B cells, contracted disease 48 days later (254 days) than WT3WT mice (206 days) at low-dose prion challenge. Delayed disease progression in CD21/35 / 3WT mice was surprising, and may point to a previously unrecognized function for B cell expression of CD21/35 in prion pathogenesis. FDCs express significant amounts of CD21/35 We attribute the impaired progression of prion disease in WT3CD21/35 / mice to their lack of CD21/35 expression on FIGURE 5. FDCs express significant amounts of CD21/35. Immunohistochemical staining of consecutive splenic sections from WT, CD21/ 35 /, and CD21/35 / 3WT chimeric mice with Abs recognizing CD21/35 and the FDC marker FDC-M1. B cells and FDCs express CD21/35 in WT (A and B), but not CD21/35 / (C and D), spleens. Although splenic B cells from CD21/35 / 3WT chimeric mice do not express CD21/35, FDCs from these mice express significant amounts of CD21/35 (E and F) relative to WT spleens. Scale bar, 50 m. G, Quantification of CD21/35 and FDC-M1 signal intensities in splenic follicles. The ratio of CD21/35:FDC-M1 staining reveals significant CD21/35 expression on B cells and FDCs in WT mice (3.1 1), none in CD21/35 / mice ( ), and CD21/35 expression exclusively on FDCs in CD21/35 / 3 WT mice ( ).

7 The Journal of Immunology 6149 FIGURE 6. PrP Sc accumulation correlates with CD21/35 expression on FDCs at 47 dpi. Consecutive sections display splenic follicles (H&E) that contain PrP Sc deposits (histoblots) and express both CD21/35 (green) and PrP C (red) in WT3WT (A) and CD21/35 / 3WT (D) mice (IF), but no PrP Sc deposition in splenic follicles lacking CD21/35 in CD21/35 / 3 CD21/35 / (B) and WT3 CD21/35 / (C) mice. Higher magnifications of the boxed areas are shown to the right of each figure. Scale bars, 200 m. FDCs. However, because CD21/35-expressing B cells closely associate with FDCs, CD21/35 expression by FDCs has been difficult to ascertain. We analyzed spleens from CD21/35 / 3WT mice to assess CD21/35 expression exclusively by FDCs. IHC confirmed that WT splenic follicles coexpressed the B cell marker B220 (data not shown), CD21/35 and the FDC marker FDC-M1 FIGURE 7. Efficient PrP Sc accumulation correlates with CD21/35 expression on FDCs. Western blot analysis of spleens from the indicated reconstitution groups 20 dpi (A and B) or47dpi(cand D) with 6 log LD 50 RML 5.0 administered i.p. (A and B) PrP Sc detection required NaPTA precipitation from 5 mg of spleen from BM chimeric mice (WT3CD21/35 /, Prnp o/o 3CD21/35 /, CD21/ 35 / 3Prnp o/o, CD21/35 / 3WT, and CD21/ 35 / 3 CD21/35 / ) unless they expressed CD21/35 on both B cells and FDCs (WT3WT, WT3Prnp o/o, and Prnp o/o 3WT mice). C, NaPTAprecipitated PrP Sc was not detected 47 dpi in spleens from Prnp o/o CD21/35 / BM chimeric mice (P / C / 3P / C / and C / 3 P / C / ) unless they were reconstituted with CD21-expressing BM (P / 3P / C / and WT / 3P / C / mice), indicating CD21/35 capture of PrP Sc. D, PrP Sc detection required NaPTA precipitation from spleens of Prnp o/o or CD21/35 / mice reconstituted with BM lacking both PrP C and CD21/35 (P / C / 3P / and P / C / 3C / mice, respectively), whose FDCs lack either PrP C or CD21/35. Expression of both CD21/35 and PrP C on FDCs (P / C / 3WT and WT3WT mice) restored efficient capture and replication of PrP Sc, which was detected without NaPTA precipitation. (Fig. 5, A and B), while CD21/35 / follicles expressed B220 (data not shown), FDC-M1 but no CD21/35 (Fig. 5, C and D). These data confirm the absence of CD21/35 on CD21/35 / B cells, the BM progenitors of which we used to reconstitute the hemopoietic compartment in lethally irradiated WT mice (CD21/ 35 / 3WT). B cells did not express CD21/35, while FDCs expressed substantial amounts of CD21/35 in CD21/35 / 3WT spleens (Fig. 5, E and F). Quantification of the ratio of CD21/35: FDC-M1 signal intensities from IHC in CD21/35 / 3WT splenic follicles (Fig. 5G) confirmed that FDC-M1 staining correlated very closely with CD21/35 ( , n 27 follicles) in splenic follicles, convincingly demonstrating significant CD21/35 expression by FDCs. The CD21/35:FDC-M1 ratio increased to in WT follicles (n 27), most likely due to B cell expression of CD21/35. The CD21/35:FDC-M1 ratio in CD21/35 / follicles is (n 29), confirming lack of CD21/35 expression. Loss of CD21/35 expression on FDCs impairs early splenic PrP Sc accumulation and replication We next investigated whether disease progression correlates with PrP Sc deposition and CD21/35 expression in reciprocal BM chimeric mice (Fig. 6). Histoblot analyses revealed significant PrP Sc deposition in WT3WT and CD21/35 / 3WT mice at 47 dpi, whose splenic follicles coexpressed PrP C and CD21/35 (Fig. 6, A and D). No PrP Sc was detected in CD21/35 / 3CD21/35 / or WT3CD21/35 / splenic follicles, which lack CD21/35 expression entirely or on FDCs, respectively (Fig. 6, B and C). Thus, among the BM chimeras, we detected PrP Sc only in spleens that express CD21/35 on FDCs, which colocally expressed CD21/35 and PrP C. We detected no PrP Sc on FDCs expressing PrP C but not CD21/35.

8 6150 CD12/35 EXPEDITES PRION NEUROINVASION CD21/35 improves PrP Sc retention To examine retention of PrP Sc by CD21/35 in more detail, we inoculated high doses of prions i.p. into reciprocal BM chimeric mice with segregated CD21/35 and PrP expression on B cells or FDCs. Twenty days after inoculation, most samples required NaPTA precipitation of PrP Sc from 5 mg of spleen for detection (Fig. 7, A and B). Eliminating CD21/35 expression on either B cells or FDCs significantly diminished PrP Sc retention (WT3CD21/35 /, Prnp o/o 3CD21/35 /,WT3CD21/35 /, CD21/35 / 3Prnp o/o, and CD21/35 / 3CD21/35 / mice). PrP Sc was detected in 50 g of spleen homogenate without NaPTA precipitation only in samples from mice expressing CD21/35 on both B cells and FDCs (WT3WT, WT3Prnp o/o and Prnp o/o 3 WT mice). Splenic PrP Sc detected at 20 dpi is most likely from the initial prion inoculum, the capture of which may be aided by CD21/35. To investigate whether early prion replication might contribute to the detected PrP Sc, we inoculated high doses of prions i.p. into BM chimeric mice lacking PrP C expression, but retaining CD21/35 expression. We crossed CD21/35 / and Prnp o/o mice, and performed reciprocal BM reconstitutions with Prnp o/o CD21/35 /, Prnp o/o, CD21/35 /, or WT mice. We then inoculated these BM chimeras i.p. with 6 log LD 50 of RML prions and analyzed their spleens for PrP Sc. Because mice lacking PrP C cannot replicate prions, any PrP Sc detected in their spleen must originate from the original inocula. At 47 dpi, we detected no PrP Sc in spleens from mice lacking both PrP C and CD21/35 (Prnp o/o CD21/35 / 3Prnp o/o CD21/35 / ). Spleens lacking PrP C expression retained PrP Sc if CD21/35 was present (Prnp o/o 3Prnp o/o CD21/35 / and Prnp o/o CD21/35 / 3Prnp o/o mice), especially on FDCs (Fig. 7, C and D). We detected PrP Sc in spleens with B cells expressing only CD21/35 (Prnp o/o 3Prnp o/o CD21/35 / mice), but not in spleens with B cells expressing only PrP C (CD21/35 / 3Prnp o/o CD21/ 35 / mice). This suggests not only that B cells retain PrP Sc, but also that they do so more efficiently when they express CD21/35 rather than PrP C. We detected significantly more PrP Sc from spleens with FDCs expressing CD21/35, PrP C, or both (Fig. 7D). Spleens incapable of prion replication that expressed CD21/35 on FDCs (Prnp o/o CD21/35 / 3Prnp o/o mice) retained PrP Sc as efficiently as replication-competent spleens that lacked CD21/35 (Prnp o/o CD21/35 / 3CD21/35 / mice). In the latter sample, however, we cannot exclude the possibility that prion replication, in addition to retention, by PrP C -expressing FDCs contributed to the PrP Sc content that we detected. Samples from spleens expressing both CD21/35 and PrP C on FDCs (Prnp o/o CD21/35 / 3WT and WT3WT mice) did not require NaPTA precipitation for detection, strongly implicating FDCs as the primary source of PrP Sc accumulation and replication. Discussion We investigated the cellular and molecular mechanisms underlying the involvement of FDCs, B cells, the CD21/35 receptors and their ligands in prion accumulation, replication, and disease progression. Simultaneous elimination of C3 and C4 extended the delay to terminal prion disease observed when only C3 was eliminated, suggesting a role for both CD21/35 ligand sources in prion propagation upon i.p. inoculation. Although mice deficient in both CD21/35 ligand sources exhibit a more significant delay than C3 / mice (46), they still succumbed to disease significantly earlier than CD21/35 / mice, a significant number of which did not contract disease at all. These data contrast with data previously reported using mice which had been erroneously reported to lack CD21/35 expression (61). In these mice, a phenomenon known as exon skipping (63) occurred that caused inappropriate splicing of exons 8 and 11, deletion of targeted exons 9 and 10, and creation of an in-frame transcript that produced CD21/35 proteins (CD21/35 hypo ) that are 14 kda smaller, yet retain domains required for CD19 interaction and ligand binding (62). CD21/35 hypo mice exhibit modestly delayed incubation times after low-dose i.p. prion inoculation (46). Mice used in the current study possess a bona fide homozygous ablation of the CD21/35 promoter and signal sequence that completely eliminates CD21/35 protein expression (54). After low-dose i.p. prion inoculation, we now found that CD21/35 / mice exhibited dramatically prolonged incubation and incomplete attack rates when compared with congenic controls. CD21/35 / mice also presented with significantly delayed prion neuropathology. The role of CD21/35 in prion pathogenesis may therefore be more prominent than previously appreciated. CD21/35 hypo and C3 / mice show similar delays in peripheral prion pathogenesis even though FDCs failed to bind measurable C3 in CD21/35 hypo mice (61, 64). C4 could compensate for lack of C3 binding on FDCs of CD21/35 hypo mice. Indeed, C3/4 / and CD21/35 / mice exhibited similar delays in prion pathogenesis when inoculated with high doses of prions. However, when inoculated with a lower prion dose, CD21/35 / mice exhibited decreased incidence and longer delay in disease progression than C3/4 / mice. Thus, CD21/35 appear capable of enhancing prion disease independent of known endogenous CD21/35 ligands, most likely by enhancing prion retention. We found substantial prion titers at very early time points in spleens from WT mice inoculated i.p. with prions, but no detectable prion titers in spleens of mice lacking CD21/35. PrP Sc correlated with CD21/35 and PrP C expression in infectious splenic follicles, presumably on FDCs, which have been convincingly shown to mediate peripheral prion pathogenesis (28 32). We hypothesized that FDCs require CD21/35 expression to maximize prion accumulation and replication. However, FDC expression of CD21/35 has been difficult to evaluate because of their intimate contact with germinal center B cells, which have confirmed CD21/35 expression. We confirmed substantial CD21/35 expression on FDCs by IHC of splenic follicles from BM chimeric mice lacking CD21/35 expression on B cells. These data corroborate FACS data from a recent report documenting CD21/35 expression on purified FDCs from lethally irradiated WT mice (65). On B cells, CD21/35 function as Ag presentation molecules and as part of an important B cell coreceptor complex with CD19 and CD81 that helps activate B cells. On FDCs, they are believed to act solely as Ag-presentation molecules. To determine its importance on each cell type during a prion infection, we restricted CD21/35 expression to either B cells or FDCs by reconstituting the hemopoietic system of lethally irradiated CD21/35 / or WT mice with BM from WT or CD21/35 / mice, respectively. FDC expression of CD21/35 was required for PrP Sc accumulation in splenic follicles early in infection, and resulted in the most expedient terminal prion disease that we observed. From these and previous data, we expected a more prominent role for CD21/35 on FDCs, which is thought to use CD21/35 mainly to trap and display Ag because they lack signaling components of the B cell coreceptor. More surprisingly, eliminating CD21/35 expression on B cells resulted in a significant delay in disease progression when compared with WT mice. Moreover, B cell-restricted CD21/35 expression abrogated the decreased incidence of terminal disease that we observed at low-dose inoculation of prions for mice completely lacking CD21/35. These data point to a previously undiscovered function in prion pathogenesis for this receptor on B cells, the primary role

9 The Journal of Immunology of which was thought to be to supply lymphotoxins to FDCs because PrP C expression occurs on only a small subset of B cells (39) and is dispensable to promote disease (43). Circulating B cells may also function as APCs, actively trapping and transporting PrP Sc from peripheral sites to lymphoid follicles, a function proposed for other circulating immune cells such as dendritic cells (66, 67) and macrophages (45, 68 70). Infectivity assays revealed no detectable prion titers in CD21/ 35 / mice even at 15 dpi, suggesting that CD21/35 facilitates prion retention on cells that express it. Using additional BM chimeric mice with segregated CD21/35 and PrP C expression on B cells or FDCs, we investigated PrP Sc retention in more detail. At 20 dpi, we discovered that the most efficient PrP Sc trapping required CD21/35 expression on both B cells and FDCs. PrP Sc detection in spleens lacking CD21/35 on either cell type required concentrating PrP Sc from 100-fold more splenic tissue. We further asked whether the PrP Sc that we detected at this very early time point was from residual inocula or nascent PrP Sc. We found severe impairment of prion retention in the absence of CD21/35 in spleens incapable of prion replication at 47 dpi. Retention improved dramatically when CD21/35 expression was restored, especially on FDCs. Surprisingly, we even detected inocula in replication-deficient spleens expressing CD21/35 on B cells, but not in spleens expressing PrP C on B cells but lacking CD21/35. We conclude that CD21/35 retains PrP Sc more efficiently than does PrP C. Although CD21/35 improved PrP Sc retention on both B cells and FDCs, retention was much more efficient by CD21/35-expressing FDCs. These data confirmed our histoblot experiments, where FDC expression of CD21/35 was required for PrP Sc accumulation. CD21/35 expression solely on B cells proved inadequate for accumulating detectable amounts of PrP Sc. Clearly, CD21/35 affected prion accumulation such that, in their absence, replication and neuroinvasion are impaired to the extent that 25% of infected mice never progress to terminal disease. We conclude that the lack of CD21/35 sufficiently slows disease kinetics to allow a significant number of mice to survive prion infection. CD21/35 may be a receptor used by FDCs for trapping prions in lymphoid follicles. Increased retention of PrP Sc by CD21/35 on FDCs could induce a persistent state of PrP Sc presentation to adjacent B cells sufficient to cause an atypical germinal center response previously reported (71). This aberrant germinal center environment may prove to be the optimal prion bioreactor. Hypertrophic FDC dendrites bearing CD21/35 loaded with PrP Sc could present aggregates to PrP C expressed on dendrites of the same cell or on neighboring cells. Although opsonization of prions by known CD21/35 ligands may mediate trapping, we conclude that it is not required. Whether (an)other unidentified protein(s) can or must mediate CD21/35- enhanced prion disease has yet to be determined. The soluble complement protein C1q is one possible candidate. C1q deficiency dramatically impaired peripheral prion pathogenesis similar to CD21/35 (46, 47) and in vitro studies have shown that C1q directly binds specific conformers of PrP C that are postulated to mimic PrP Sc (48). More recently, C1q binding has also been shown to depend on PrP C density (72). C1q also depleted complement activation through the classical pathway and fixed C4 in a hemolytic assay. Given these data and ours, C1q appears more likely to mediate a possible interaction between PrP Sc and CD21/35 than C4. C1q has been reported to be a ligand for human CD35 (73), although this has yet to be confirmed in mice. Another possibility is mannan-binding lectin, a homolog of C1q that can also activate complement through the classical pathway. Mannan-binding lectin has been shown to bind human CD35 (74, 75), although direct binding to PrP Sc was not detected (72). Alternatively, CD21/35 may bind prions directly to mediate accumulation, replication, and neuroinvasion. Experiments designed to test this idea may uncover the molecular basis for extraneural prion infections. Acknowledgments We thank Denis Marino for help with the BM reconstitutions and Ed Hoover and Anne Avery for critical reading of the manuscript. Disclosures The authors have no financial conflict of interest References 1. Baker, H. F., and R. M. Ridley What went wrong in Bse from prion disease to public disaster. Brain Res. Bull. 40: Will, R. G., J. W. Ironside, M. Zeidler, S. N. Cousens, K. Estibeiro, A. Alperovitch, S. Poser, M. Pocchiari, A. Hofman, and P. G. 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