1

BAFFR deficiency results in limited CD169+ macrophage function during viral infection 1

2

Haifeng C. Xu1,2,3,#, Jun Huang2,#, Vishal Khairnar3, Vikas Duhan3, Aleksandra A. Pandyra3 , Melanie 3

Grusdat2, Prashant Shinde2, David R. McIlwain2,4, Sathish Kumar Maney2, Jennifer Gommerman5, Max 4

Löhning6,7, Pamela S. Ohashi1,5, Tak W. Mak1,5, Kathrin Pieper8, Heiko Sic8, Matthaios Speletas9, 5

Hermann Eibel8, Carl F. Ware10, Alexei V. Tumanov11, Andrey A. Kruglov6, Sergei A. Nedospasov6,14, 6

Dieter Häusinger2, Mike Recher12*, Karl S. Lang3*, and Philipp A. Lang1,2,13* 7

8 1Campell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University 9 Health Network (UHN), 620 University Ave., Toronto, Ontario, Canada M5G 2C1 10 2Department of Gastroenterology, Hepatology and Infectious Diseases, University of Düsseldorf, 11 Universitätsstr. 1, 40225 Düsseldorf, Germany 12 3Institute of Immunology, Medical Faculty, University of Duisburg-Essen, Hufelandstr. 55, Essen 13 45147, Germany 14 4Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford 15 University, Stanford, California, 94305, USA 16 5Department of Immunology, University of Toronto, 1 King´s Circle, Toronto, Ontario Canada, M5S 17 1A8 18 6Experimental Immunology, Department of Rheumatology and Clinical Immunology, Charité University 19 Medicine Berlin, 10117 Berlin, Germany 20 7German Rheumatism Research Center (DRFZ), a Leibniz Institute, 10117 Berlin, Germany 21 8University Medical Centre Freiburg, Center of Chronic Immunodeficiency, Engesserstraße 4, D-79110 22 Freiburg, Germany 23 9Department of Immunology & Histocompatibility, Faculty of Medicine, University of Thessaly, Larissa, 24 Greece 25 10Sanford Burnham Medical Research Institute, 10901, La Jolla, CA 92037, USA 26 11Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983 27 12Primary Immunodeficiency Clinic, Medical Outpatient Division and Immunodeficiency Lab, 28 Department of Biomedicine, University Hospital Basel, Switzerland 29 13Department of Molecular Medicine II, Universitatsstrasse 1, D-40225 Düsseldorf, Germany 30 14Lomonosov Moscow State University, Moscow 119991 Russia. 31 32 #,* contributed equally to this work 33 34 35 Address correspondence to Philipp A. Lang MD, PhD, Department of Gastroenterology, Hepatology 36

and Infectious Diseases, Universitätsstrasse 1, Geb. 23.12.U1 Room 41, D-40225 Düsseldorf, 37

Germany, tel.: +49 211 8113580, fax.: +49 211 8113421, Philipp.Lang@med.uni-duesseldorf.de 38

JVI Accepted Manuscript Posted Online 11 February 2015J. Virol. doi:10.1128/JVI.02976-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.

2

Abstract 39

The B cell activating factor of the TNF family (BAFF) is critical for B-cell development and humoral 40

immunity in mice and humans. While the role of BAFF in B cells has been widely described, its role in 41

innate immunity remains unknown. Using BAFFR deficient mice we characterized BAFFR related 42

innate and adaptive immune functions following infection with vesicular stomatitis virus (VSV) and 43

lymphocytic choriomeningitis virus (LCMV). We identified a critical role for BAFFR signalling in the 44

generation and maintenance of the CD169+ macrophage compartment. Consequently, Baffr–/– mice 45

exhibited limited induction of innate type I interferon production after viral infection. Lack of BAFFR 46

signalling reduced virus amplification and presentation following viral infection, resulting in highly 47

reduced anti-viral adaptive immune responses. As a consequence, BAFFR deficient mice showed 48

exacerbated and fatal disease after viral infection. Mechanistically, transient lack of B cells in Baffr–/– 49

animals resulted in limited lymphotoxin expression, which was critical for maintenance of CD169+ cells. 50

In conclusion, BAFFR signalling affects both innate and adaptive immune activation during viral 51

infections. 52

53

Importance 54

Viruses cause acute and chronic infections in humans resulting in millions of deaths every year. Innate 55

immunity is critical for the outcome of a viral infection. Innate type I interferon production can limit viral 56

replication while adaptive immune priming by innate immune cells induces pathogen specific immunity 57

with long term protection. Here we show that BAFFR deficiency not only perturbed B cells, but also 58

resulted in limited CD169+ macrophages. These macrophages are critical in amplifying viral particles 59

to trigger type I interferon production and initiate adaptive immune priming. Consequently, BAFFR 60

deficiency resulted in reduced enforced viral replication, limited type I interferon production, and 61

reduced adaptive immunity when compared to BAFFR competent controls. As a result, BAFFR 62

deficient mice were predisposed to fatal viral infections. Thus, BAFFR expression is critical for innate 63

immune activation and anti-viral immunity. 64

3

Introduction 65

The B cell activating factor of the TNF family (BAFF) binding receptor (BAFFR) is critical for B 66

cell development(1, 2). Patients lacking the BAFFR have been identified within cohorts with common 67

variable immunodeficiency, the most prevalent symptomatic primary immunodeficiency in adult 68

patients. BAFFR deficient humans exhibit severe B cell lymphopenia and impaired immunglobulin 69

production(3). Similarly, the lack of BAFF signalling in Baffr–/– mice is also associated with severe B 70

cell lymphopenia(4). BAFF binds to three receptors, the BAFF receptor (BAFFR), transmembrane 71

activator and CAML interactor (TACI), and B cell maturation antigen (BCMA)(5). While TACI and 72

BCMA engage both BAFF and a proliferation inducing ligand (APRIL), BAFFR binds BAFF exclusively. 73

The BAFF – BAFFR association leads to recruitment and degradation of TRAF3(6). TRAF3 negatively 74

regulates NF-NB-inducing kinase (NIK), the upstream kinase for NF-NB2 activation(7). In the presence 75

of BAFF, degradation of TRAF3 leads to stabilization of NIK and activation NF-NB2, which triggers B 76

cell survival(8-11). Furthermore, BAFF triggers activation of Akt signalling pathways, which increase 77

the metabolic activity of B cells(12). Additionally, BAFF transmits survival signals via Erk activation, 78

which triggers phosphorylation and degradation of the pro-apoptotic molecule Bim(13-15). Together, 79

these signalling pathways promote BAFF-mediated survival of B cells. Whether innate immunity may 80

be abnormal in Baffr–/– mice has not yet been investigated. 81

Successful defense against viral infections relies on effective innate and adaptive immunity. 82

During infection, viral particles are captured in secondary lymphoid organs such as the lymph node or 83

the spleen(16). In mice, macrophages in the marginal sinus in the splenic white pulp or macrophages 84

located in the subcapsular space of the lymph node filter pathogens from the blood and the lymph 85

respectively(16-18). Metallophilic macrophages and subcapsular sinus macrophages are 86

characterized by expression of the C-type lectin CD169 (Siglec-1)(19). CD169+ cells are critical for 87

innate cytokine production and viral antigen presentation to B cells and represent an important link 88

between innate and adaptive immunity(18). In particular, Usp18 driven suppression of anti-viral type I 89

interferon signalling allows viral replication to occur in CD169+ cells in close proximity to marginal zone 90

B cells(20, 21). Consequently, large quantities of viral antigen are made available in order to induce 91

rapid and robust adaptive immunity(22-25). The rapid induction of adaptive immune responses as a 92

result of early viral replication in the spleen guarantees virus elimination and survival of the virus 93

infected host(20, 21). 94

4

B cells are important for the generation of the splenic architecture including the maintenance 95

of the marginal zone(26, 27). Following a recent report indicating that BAFF is produced by neutrophils 96

in the marginal zone of the spleen(28), we chose to investigate the impact of BAFFR signalling on 97

innate immune responses. We found that absence of BAFFR resulted in reduced lymphotoxin 98

expression, decreased presence of CD169+ cells, delayed and impaired innate and adaptive immune 99

activation, and consequently promotion of fatal disease development after viral infection. 100

5

Materials and Methods: 101

Mice: Mice were infected intravenously with VSV or LCMV at the indicated doses. Baffr–/– mice were 102

bred on a C57BL/6 genetic background(4). CD45.1+ mice were purchased from Jackson Laboratory. 103

Ltbfl/fl x CD19-Cre+ and Ifnar1-/- mice were previously described(29, 30). All experiments were 104

performed in single ventilated cages. During survival experiments, the health status of the mice was 105

checked twice daily. Upon the appearance of clinical signs of VSV replication in the central nervous 106

system (CNS), such as paralysis, mice were removed from the experiment. Animal experiments were 107

carried out with the authorization of the Veterinäramt of Nordrhein Westfalen, Germany, in accordance 108

with the German law for animal protection and the institutional guidelines of the Ontario Cancer 109

Institute. 110

111

Viruses: VSV, Indiana strain (VSV-IND, Mudd-Summers isolate), was originally obtained from Prof. D. 112

Kolakofsky (University of Geneva, Switzerland). Virus was propagated on BHK-21 cells at a multiplicity 113

of infection (MOI) of 0.01 and was then plaqued onto Vero cells. LCMV was used and virus titers 114

determined as previously described(21). Viruses were administrated to mice through intravenous 115

injection. 116

117

Poly I:C and recombinant interferon: poly I:C (GE Healthcare Life Sciences) and mouse 118

recombinant interferon alpha A (PBL Biosciences) were administrated to mice through intravenous 119

injection at indicated doses. 120

121

Neutralizing antibodies were determined by plaque reduction neutralization test (PRNT) as 122

previously described(21). Serum was prediluted (1:40). Complement system was inactivated (56°C for 123

30 min). Serum was titrated 1:2 over 12 steps and incubated with 1000 PFU of VSV. After 90 minutes 124

of incubation, the virus-serum mixture was plaqued on Vero cells. Overlay was added after one hour. 125

Plaques were counted 24 hours later by crystal violet staining. The cut of was 50% reduction plaques 126

when compare to serum free controls 127

128

ELISA: Interferon alpha ELISA (PBL Biosciences) was performed as instructed by the manufacturer. 129

130

6

Purification of B cells: For B cell purification, single cell suspended splenocytes were enriched 131

following the manufacturer’s instructions with the CD45R (B220) MicroBeads, mouse kit (Miltenyi) 132

133

RT-PCR analyses: RNA purification and RT-PCR analyses were performed as previously described 134

according to manufacturer’s instructions (Qiagen)(31). Gene expression of Isg15, Mx1, Ifit2, Lta, Ltb 135

and Gapdh was performed using kits from Applied Biosystems. For analysis, the expression levels of 136

all target genes were normalized to Gapdh expression levels (∆Ct). Gene expression values were then 137

calculated based on the ∆∆Ct method relative to naïve WT controls. Relative quantities (RQ) were 138

determined using the equation: RQ=2^-∆∆Ct. 139

140

Histology: Histological analyses were performed on snap-frozen tissue as described previously by 141

using self-made anti-VSV-G monoclonal antibody (clone Vi10)(21). CD90.2, CD4, CD8 and B220 142

antibody was purchased from eBioscience (San Diego, CA). CD169 (clone: MOMA-1) antibody was 143

obtained from Abcam (Cambridge, MA). H&E staining was previously described(32). Bars indicate 50 144

μm or 100 µm, as indicated in the corresponding figure legend. 145

146

Flow cytometry: Analyses were performed such as previously described for LCMV tetramer staining 147

and intracellular cytokine staining(33). Different spleen immune populations were identified using anti-148

B220 (RA3-6B2), anti-MHCII (M5/114.15.2), anti-CD11c (N418), anti-GR-1 (RB6-8C5), anti-F4/80 149

(BM8), anti-CD3 (145-2C11), anti-CD8 (52-6.7), anti-CD4 (GK1.5) antibodies (clone). Stem cell 150

analyses were performed as previously described(34). Lin– was considered negative staining for: CD3, 151

CD11b (M1/70), Ly6C, Ter119 (TER-119), CD19, CD11c, MHCII, IL7R (A7R34), and NK1.1. CMP and 152

GMP were determined using anti-CD34 (RAM34), anti-CD16/32 (93), anti-Sca1 (D7), and anti-CD117 153

(2B8) antibodies. All antibodies were obtained from eBioscience (San Diego CA), except anti-CD169 154

(3D6.112), which was obtained from AbD Serotec (Dusseldorf). 155

156

Statistical analysis: Data were expressed as means ± S.E.M. Student’s t-test was used to detect 157

statistically significant differences between two groups. Significant differences between several groups 158

were detected by one-way analysis of variance (ANOVA) with Bonferroni or Dunnett post hoc tests or 159

mentioned specifically in the corresponding figure legend. The level of statistical significance was set 160

at P < 0.05. 161

7

Results 162

BAFFR is critical in overcoming viral infection. 163

Murine BAFFR deficiency resulted in severe B cell lymphopenia but did not have a major 164

impact on T cell, dendritic cell or neutrophil numbers (FIG 1A-B). As expected, Baffr–/– mice exhibited 165

delayed virus neutralizing antibody production after infection with the cytolytic vesicular stomatitis virus 166

(VSV) (FIG 1C). Consistent with a requirement for rapid neutralizing antibody formation for elimination 167

of VSV(21), Baffr–/– mice succumbed to VSV infection, while wild-type (WT) animals overcame the 168

infection (FIG 1D). When neuronal tissue was harvested from the sick animals (day 7-10), the 169

presence of VSV could be detected (data not shown). These data suggest that absence of BAFFR 170

resulted in fatal disease development during infection with VSV. 171

172

BAFFR mediates enforced viral replication during viral infection 173

Despite detection of VSV replication in the CNS in the later phase of infection, VSV titers were 174

below detection limit in spleen tissue of Baffr-/- animals 8h and 24h after infection with VSV, while virus 175

was readily detectable in WT controls (FIG 2A). Overcoming VSV infection is not only dependent on 176

production of neutralizing antibodies, but also on innate type I interferon production(30, 35, 36). 177

Consistent with decreased replication of VSV in spleen tissue, Baffr–/– mice had reduced type I 178

interferon levels in the serum when compared to infected WT mice (Figure 2B). Moreover, the 179

increase of interferon regulated genes (IRGs) such as Isg15, Mx1, and Ifit2 24 hours after VSV 180

infection was reduced in brain tissue harvested from BAFFR deficient animals when compared to their 181

corresponding controls (FIG 2C). However, competent production of type I interferon was detectable in 182

Baffr–/– mice following injection of the TLR3 agonist poly I:C in vivo (FIG 2D). Furthermore, IRG 183

expression in the brain tissue from Baffr-/- mice and their WT controls following treatment with poly I:C 184

was similar (FIG 2E). This indicates that the capacity to induce type I interferon was normal in Baffr–/– 185

mice. Moreover when animals were treated with mouse recombinant IFN-α, similar brain IRG 186

expression was observed between BAFFR deficient and WT mice (FIG 2E). Collectively, reduced type 187

I interferon levels in Baffr–/– mice following virus infection, was likely explainable by a different 188

mechanism than defective pathogen recognition receptor (PRR) signalling. 189

190

BAFF signalling is required for maintenance of metallophilic macrophages in the spleen. 191

8

We have recently demonstrated that early virus replication in the spleen depends on CD169+ 192

metallophilic macrophages and is triggered by Usp18 mediated resistance to type I interferon in these 193

cells(21). In mice, CD169+ cells in the spleen are in direct contact with the blood stream and remove 194

virus particles and apoptotic cells from circulation(21, 37). Lack of CD169+ macrophages blocks virus 195

replication early after infection, causing limited antigen amplification and reduced virus induced 196

immune activation(21, 38). Since BAFFR deficient animals exhibited lower viral replication during early 197

infection, we investigated potential mechanisms by which BAFFR signalling might control enforced 198

viral replication. Myeloid progenitor populations in the bone marrow such as the common myeloid 199

progenitors (CMP: Lin–, Sca1–, IL7R–, CD117+, CD34+, CD16/32–) or the granulocyte and macrophage 200

progenitors (GMP: Lin–, Sca1–, IL7 R–, CD117+, CD34+, CD16/32+) did not differ between Baffr–/– and 201

WT mice (FIG 3A). However, CD169+ metallophilic macrophages which have a critical role during 202

early viral replication(21), were highly reduced in spleen tissue of Baffr–/– mice compared to WT 203

animals (FIG 3B), while red pulp macrophages were present at similar frequencies (FIG 3C). Taken 204

together, BAFFR appears to be required for maintenance of CD169+ cells. 205

Next, we investigated the presence of CD169+ cells following infection. CD169+ cells are 206

present at reduced numbers in BAFFR deficient animals, but a residual population is still detectable in 207

the naïve state (FIG 3D-E). However, shortly after infection with VSV, CD169+ cells rapidly disappear 208

in Baffr–/– mice in contrast to WT animals (FIG 3D). As a consequence of reduced CD169+ 209

macrophages, VSV protein (detected by the VSV-specific monoclonal antibody Vi10) was reduced in 210

Baffr–/– mice in spleen sections following VSV infection, while it was readily detectable in spleen 211

sections from WT animals (FIG 3E). Next, we wondered whether type I interferon directly affected 212

CD169+ cell survival. However, the distribution of CD169+ cells in spleen tissues harvested from 213

Ifnar1–/– mice and WT animals was similar (FIG 4). Taken together, these data indicate that lack of 214

BAFFR expression is associated with a decreased presence of splenic CD169+ cells after virus 215

infection, which consequently diminishes early viral replication. 216

217

BAFFR deficiency results in reduced B cell mediated maintenance of CD169+ cells. 218

Next, we addressed whether the reduction of CD169+ cells in Baffr–/– mice occurred due to 219

severe B cell lymphopenia(39, 40). We transferred WT B cells into Baffr–/– mice and monitored 220

CD169+ cells in spleen tissue after 40 days. Interestingly, CD169+ cells were readily detectable in 221

spleen tissue of BAFFR deficient mice that were supplemented with B cells (FIG 5A). Furthermore, 222

9

type I interferon production following VSV infection could be partially rescued by the transfer of WT B 223

cells into Baffr–/– mice (FIG 5B). Moreover, neutralizing antibody titers of the Baffr–/– animals which 224

received WT B cells was increased when compared into Baffr–/– animals (FIG 5C), and animals could 225

overcome the VSV infection (FIG 5D). These data indicate that B cells mediate maintenance of 226

CD169+ cells and consequently contribute to innate immunity during infection. 227

Lymphotoxin signalling is critical for CD169+ cell development in the spleen and lymph node 228

tissue(32, 38, 39, 41). Moreover, it has been shown that lymphotoxins derived from B cells which are 229

important for maintenance of CD169+ cells(29, 39). Consistently, BAFFR deficient animals exhibited 230

lower lymphotoxin alpha and lymphotoxin beta expression levels compared to their corresponding 231

controls (FIG 6A). These data suggest that impaired B cell numbers in Baffr–/– mice may contribute to 232

insufficient lymphotoxin expression to maintain normal levels of CD169+ cells(29, 38, 41, 42). To 233

investigate the role of lymphotoxin beta during enforced viral replication, we infected Ltbfl/flxCD19-Cre+ 234

animals and compared them to their corresponding controls. As expected, these animals exhibited 235

less CD169+ cells than the WT controls (FIG 6B) (29, 42). Consistent with previous reports and our 236

data obtained in Baffr–/– mice, we observed reduced type I interferon production shortly after infection 237

in Ltbfl/flxCD19-Cre+ when compared to Ltbfl/flxCD19-Cre- animals (FIG 6C) (39). Furthermore, low dose 238

infection resulted in reduced production of neutralizing antibodies (FIG 6D). These data indicate that 239

lack of B cells or B cell derived expression of LtE results in limited innate immune activation and 240

delayed adaptive immune priming. 241

242

BAFFR deficiency results in limited innate immune activation following LCMV infection. 243

To further analyze the importance of CD169+ macrophages in viral replication and the 244

induction of anti-viral immunity, we examined Baffr–/– mice following LCMV infection. Consistent with 245

the VSV infection, we observed reduced LCMV replication in the spleen tissue of Baffr-/- mice in 246

comparison to WT controls at 72 hours after infection (FIG 7A). Furthermore type I interferon levels 247

were highly reduced in the serum of Baffr-/- animals when compared to WT controls (FIG 7B). 248

Tetramer+ LCMV specific T cells were highly reduced following LCMV infection of Baffr–/– mice when 249

compared to WT mice (FIG 7C). Moreover, IFN-J production after in vitro re-stimulation with the 250

immunodominant LCMV peptides was reduced in both CD8+ and CD4+ T cells harvested from Baffr–/– 251

mice in comparison to WT controls (FIG 7D). When viral titers were determined 20 days after infection, 252

WT animals had eliminated the virus from all organs tested (FIG 7E). In sharp contrast, LCMV infected 253

10

Baffr–/– mice displayed high virus titers in all organs tested (FIG 7E). Collectively, these data suggest 254

that the absence of BAFFR signalling causes impaired generation of the marginal zone compartment, 255

and impaired induction of innate and adaptive immune responses during viral infection. 256

11

Discussion 257

In this study, we have identified a critical role for BAFFR in the maintenance of CD169+ 258

macrophages. Baffr–/– mice showed limited innate immune activation and reduced adaptive immune 259

priming associated with fatal disease outcome. Mechanistically, impaired B-cell development in Baffr–/– 260

mice resulted in limited lymphotoxin expression and likely as a consequence, reduced presence of 261

CD169+ cells. 262

BAFF can be produced by a variety of immune cells including dendritic cells, macrophages 263

and neutrophils(5). Interestingly, a recent report identified BAFF producing neutrophils to be located in 264

the marginal zone of the spleen(28). These neutrophil B helper cells contribute to marginal zone B cell 265

activation and antibody production against pathogens(28). Based on our results, BAFF production by 266

neutrophil B helper cells may, through promoting B cell mediated lymphotoxin production, also affect 267

CD169+ cell survival and subsequently enforce antigen amplification and presentation. Furthermore, 268

BAFF overexpression has been linked to a variety of autoimmune diseases such as rheumatoid 269

arthritis, lupus erythematosus and Sjögren syndrome(5, 43-45). A clinically used BAFF blocking 270

antibody, belimumab, is effective in treating some lupus patients (46) and potentially, some clinical 271

efficacy of BAFF neutralization in lupus patients may be due to effects on CD169+ macrophages. 272

Viral infections are potent activators of the immune system and can trigger autoimmunity(47) 273

through several mechanisms including molecular mimicry and bystander activation(48). Increased 274

BAFF levels may not only affect B cell mediated autoimmunity, but also B cell mediated effects on 275

CD169+ macrophages to increase bystander activation. Furthermore, replication of low affinity 276

antigens in CD169+ macrophages may contribute to development of virus mediated autoimmunity 277

induced by molecular mimicry(49). Considering our data, altered BAFF expression levels may lead to 278

increased immune responses during viral infections. These mechanisms could also contribute to 279

induction of autoantibodies observed during viral infections(50, 51). 280

As we show here, defects in BAFFR expression may limit innate immunity during infection. 281

This may be triggered by reduced lymphotoxin beta production of B cells, as lack of lymphotoxin beta 282

resulted in reduced presence of CD169+ macrophages (29, 38, 39). Furthermore, lymphotoxins trigger 283

innate type I interferon production during viral infection(52, 53). This may be in part triggered by 284

enforced viral replication in CD169+ cells, which is also critical for induction of adaptive immune 285

priming(21, 49). Considering our data, deletions in BAFFR may not only affect B cell driven immunity 286

12

but also trigger defects in innate immunity. Future studies may analyze the role of BAFFR deficiency 287

during innate immunity in human patients. 288

In conclusion we have identified that BAFFR deficiency mediates reduced presence of B cells 289

impacting the maintenance of CD169+ macrophages and innate immunity. 290

13

Acknowledgements: 291

The authors are grateful for the technical assistance of Eugene Bäcker, Konstanze Schättel, and 292

Stefanie Münch. MR holds a professorship from the Swiss National Science Foundation 293

(PP00P3_144863). This study was supported by the CIHR grant of Pamela S. Ohashi (CIHR- MOP-294

106529). PSO holds a Canada Research Chair in Autoimmunity and Tumor Immunity. Also, this study 295

was supported by a grant to JG (CIHR-MOP-67157). This research was funded in part by the Ontario 296

Ministry of Health and Long Term Care. The views expressed do not necessarily reflect those of the 297

OMOHLTC. Moreover, this study was supported by the Alexander von Humboldt Foundation 298

(SKA2008 and SKA2010), the German Research Council (CRC974, and LA2558/3-1/4-1/5-1). 299

14

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493 494

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Figure Legends: 495

FIG 1: BAFFR is essential for anti-viral immunity. 496

(A) Sections from snap frozen spleen tissue of WT and Baffr–/– mice were stained with H&E (scale bar 497

= 100µm) or stained with anti-CD4, anti-CD8, anti-B220 antibodies (scale bar = 50µm). (One 498

representative out of n=5 is shown). (B) Single cell suspensions from splenocytes derived from WT 499

and BAFFR deficient mice were analyzed for immune cell populations with monoclonal antibodies 500

specific for the indicated markers (n=6, n.s. indicates not significant; except for B220+ cells, % is 501

related to B220- splenocytes). (C-D) WT and Baffr–/– mice were infected with 105 pfu of VSV. (C) 502

Serum was taken at the indicated time points and analyzed for anti-viral neutralizing antibodies by 503

PRNT assay (starting with n=8). (D) Survival was monitored over the indicated time period (starting 504

with n=5). Error bars show SEM, n.s. indicates not significant. 505

506

FIG 2: BAFFR mediates enforced viral replication during viral infection 507

(A-C) WT and Baffr–/– mice were infected with 105 pfu of VSV. (A) Virus titers were measured in the 508

spleen 8h (left panel) and 24h (right panel) after infection (n=6) (B) IFN-D concentration was measured 509

12 h and 24 h after infection with 105 pfu of VSV in the serum of WT and BAFFR deficient mice (n=6). 510

(C) Isg15, Ifit2 and Mx1 mRNA expression was determined from brain tissue of infected WT and Baffr–511 /– mice after 24h p.i. (n=5). (D) IFN-D concentration was examined in the serum of WT and Baffr–/– 512

mice 3h after challenge with 25µg or 100µg poly I:C (n=6). (E) Isg15, Ifit2 and Mx1 mRNA expression 513

was determined from brain tissue of WT and Baffr-/- 6 hours after treatment of 10,000 units of mouse 514

recombinant IFN-α or 100 µg poly I:C (n=6). Error bars show SEM, n.s. indicates not significant. 515

516

FIG 3: BAFFR signals are critical for maintenance of CD169+ cells and viral replication in spleen 517

tissue early after infection. 518

(A) CMP and GMP in the bone marrow of WT and Baffr–/– mice were analyzed by flow cytometry (n=6, 519

n.s. indicates not significant). (B) Left panel, sections of snap frozen spleen tissue of WT and Baffr–/– 520

mice were stained with anti-CD169 and analyzed by fluorescence microscopy (clone: MOMA-1, one 521

representative of n=6 is shown, scale bar = 100µm). Right panel, CD169+ cells were measured in 522

splenocytes of WT vs. Baffr–/– mice by flow cytometry (n=6). (C) F4/80+ cells were analyzed in spleen 523

tissue from WT and BAFFR deficient animals by flow cytometry (n=6). (D) Snap frozen spleen 524

sections were stained with an anti-CD169 antibody 0, 3, 5, and 7 h after VSV infection of WT vs. 525

19

BAFFR deficient mice (One representative of n=6 is shown, scale bar = 100µm). (E) Sections from 526

snap frozen spleen tissue obtained from WT and Baffr–/– mice after the indicated time periods following 527

VSV infection were stained with anti-CD169 and anti-VSV-G-protein (clone: Vi10) (one representative 528

of n=6 is shown, scale bar = 100µm). 529

530

FIG 4: CD169+ cell survival following VSV infection in Ifnar1–/– mice. 531

Sections from snap frozen spleen tissue obtained from WT and Ifnar1–/– mice after the indicated time 532

periods following VSV infection were stained with anti-CD169 and anti-VSV-G-protein (clone: Vi10) 533

(one representative of n=6 is shown, scale bar = 100µm). 534

535

FIG 5: B cell dependent maintenance of CD169+ cells. 536

(A) Baffr–/– mice were reconstituted with sorted WT B cells. 40 days later, spleen tissue was harvested 537

and compared to WT and Baffr–/– mice. CD169+ (upper panels) and transferred B cells (CD45.1, 538

middle panels) are shown in WT, Baffr–/– and B cell transferred Baffr–/– mice (n=5, one representative 539

is shown, scale bars = 100µm). (B-D) Baffr–/– mice were reconstituted with sorted WT B cells. 40 days 540

later, animals were infected with 105 pfu of VSV and compared to WT and Baffr–/– mice. (B) IFN-D 541

concentration was measured 24h after infection in WT, Baffr–/– mice and WT B cell transferred Baffr–/–542

(* (p<0.05), **** (p<0.0001), Holm-Sidak test was used for post hoc test). (C) Neutralizing Ig titers 543

were determined at indicated time points after infection (n=4-5, *** (p<0.001) between WT and Baffr–/– 544

mice, ## (p<0.01), between WT and Baffr–/– mice after WT B cell transfer, §§§ (p<0.001) between 545

Baffr–/– and B cell transferred Baffr–/– mice). (D) Survival was monitored in WT, Baffr–/– mice and Baffr–546 /– mice 40 days after WT B cell transfer following infection with 105 pfu of VSV (n=7-8). Error bars 547

show SEM, n.s. indicates not significant. 548

549

FIG 6: B cell dependent maintenance of CD169+ cells depend on lymphotoxin beta expression. 550

(A) Lymphotoxin alpha (left panel) and beta (right panel) mRNA expression levels were analyzed from 551

spleen tissue harvested from WT and Baffr–/– mice (n=5-7). (B) Snap frozen spleen sections from Ltbfl/fl 552

x CD19-Cre+ mice and control animals were stained with anti-CD169 and F4/80 antibody (one 553

representative of n=3 is shown). (C) IFN-D concentration was determined 24 h after infection with 105 554

pfu of VSV from Ltbfl/fl x CD19-Cre+ and control animals (n=4-6). (D) Neutralizing antibody titers were 555

20

measured in sera harvested from Ltbfl/fl x CD19-Cre+ and control animals at the indicated time points 556

after infection (n=4-6). Error bars show SEM, n.s. indicates not significant. 557

558

FIG 7: Impaired innate and adaptive immunity in BAFFR deficient mice during LCMV infection. 559

WT and Baffr–/– were infected with 200 pfu of LCMV-Docile. (A) Virus titers were measured in spleen, 560

liver, lung, kidney, brain and spinal cord tissue 72 hours after infection (n=5-6). (B) IFN-D 561

concentration was determined in the serum of infected animals 72 hours following infection (n=5-6). (C) 562

20 days after infection virus specific CD8+ T cells were examined by tetramer staining in spleen tissue 563

of mice (n=5–6). (D) IFN-J production of T cells as assessed by intracellular cytokine staining and flow 564

cytromteric analysis was measured after in vitro re-stimulation with the MHCI peptides gp33 and 565

np396 (left panel) and the MHCII peptide gp61 (right panel) (n=5-6). (E) Virus titers were measured in 566

spleen, liver, lung, kidney, brain and spinal cord tissue 20 days after infection (n=5–6). Error bars 567

show SEM, n.s. indicates not significant. 568