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Clement, Mathew, Marsden, Morgan, Stacey, Maria, Abdul-Karim, Juneid, Gimeno Brias, Silvia,

Costa Bento, Diana Filipa, Scurr, Martin, Weaver, Casey, Carlesso, Gianluca, Clare, Simon, Jones,

Simon, Godkin, Andrew, Ghazal, Peter, Jones, Gareth and Humphreys, Ian Cytomegalovirus-

specific IL-10-producing CD4+ T cells are governed by type-I IFN-induced IL-27 and promote

virus persistence. Plos Pathogens file

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1

Cytomegalovirus-specific IL-10-producing CD4+ T cells are 1

governed by type-I IFN-induced IL-27 and promote virus 2

persistence 3

4

5

Mathew Clement1*, Morgan Marsden1, Maria A. Stacey1, Juneid Abdul-Karim1, Silvia 6

Gimeno Brias1, Diana Costa Bento1, Martin J. Scurr1, Casey T. Weaver3, Gianluca 7

Carlesso4, Simon Clare5, Simon A. Jones1, Andrew Godkin1, Peter Ghazal2, Gareth 8

W. Jones1 & Ian R. Humphreys1,5* 9

10

1Division of Infection & Immunity, Cardiff University, Cardiff, United Kingdom 11

2Division of Pathway Medicine and Edinburgh Infectious Diseases, University of 12

Edinburgh, Edinburgh, United Kingdom 13

3Department of Pathology, University of Alabama at Birmingham, Birmingham, USA 14

4Respiratory, Inflammation and Autoimmunity, Research Department, MedImmune 15

LLC, Gaithersburg, MD, USA 16

5Wellcome Trust Sanger Institute, Cambridgeshire, UK 17

18

Keywords: cytomegalovirus, CD4+ T-cells, IL-10, IL-27R, type-I IFN 19

*Corresponding authors: Dr. Mathew Clement ([email protected]) & Dr Ian R. 20

Humphreys ([email protected]) 21

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2

Abstract 28

CD4+ T cells support host defence against herpesviruses and other viral pathogens. 29

We identified that CD4+ T cells from systemic and mucosal tissues of hosts infected 30

with the β-herpesviridae human cytomegalovirus (HCMV) or murine cytomegalovirus 31

(MCMV) express the regulatory cytokine interleukin (IL)-10. IL-10+CD4+ T cells co-32

expressed TH1-associated transcription factors and chemokine receptors. Mice 33

lacking T cell-derived IL-10 elicited enhanced antiviral T cell responses and restricted 34

MCMV persistence in salivary glands and secretion in saliva. Thus, IL-10+CD4+ T 35

cells suppress antiviral immune responses against CMV. Expansion of this T-cell 36

population in the periphery was promoted by IL-27 whereas mucosal IL-10+ T cell 37

responses were ICOS-dependent. Infected Il27rα-deficient mice with reduced 38

peripheral IL-10+CD4+ T cell accumulation displayed robust T cell responses and 39

restricted MCMV persistence and shedding. Temporal inhibition experiments 40

revealed that IL-27R signaling during initial infection was required for the suppression 41

of T cell immunity and control of virus shedding during MCMV persistence. IL-27 42

production was promoted by type-I IFN, suggesting that β-herpesviridae exploit the 43

immune-regulatory properties of this antiviral pathway to establish chronicity. Further, 44

our data reveal that cytokine signaling events during initial infection profoundly 45

influence virus chronicity. 46

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3

Author summary 56

Viruses including the pathogenic β-herpesvirus human cytomegalovirus (HCMV) can 57

replicate within and disseminate from mucosal tissues. Understanding how to 58

improve antiviral immune responses to restrict virus replication in the mucosa could 59

help counter virus transmission. Studies in the murine cytomegalovirus (MCMV) 60

model have demonstrated the importance of the CD4+ T cells in control of mucosal 61

MCMV replication. However, this process is inefficient, allowing virus persistence. 62

Herein, we reveal that production by CD4+ T cells of the immune-suppressive soluble 63

protein, or cytokine, interleukin (IL)-10 facilitates virus persistence in mucosal tissue. 64

Mice deficient in T cell-derived IL-10 mounted heightened T cell responses and 65

reduced virus replication in the salivary glands and shedding in the saliva. The 66

cytokine IL-27 induced IL-10-producing CD4+ T cells in the periphery whereas a cell 67

surface-expressed protein, ICOS, promoted mucosal IL-10+ T cell responses. IL-27 68

acted in the initial stages of infection to impinge on T cell responses and antiviral 69

control. In turn, IL-27 production in response to viral infection was triggered by type I 70

interferon, a prototypic antiviral cytokine. Thus, our data suggest that herpesviruses 71

may exploit immune-suppressive properties of this early antiviral cytokine response 72

to facilitate persistence within and shedding from mucosal tissue. 73

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4

Introduction 84

85

Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus that establishes 86

lifelong infection. Infectious virus is usually acquired by horizontal transmission via 87

mucosal secretions and urine. HCMV infection is typically asymptomatic in healthy 88

individuals. However in the immunocompromised such as HIV-infected individuals 89

and patients receiving immune-suppressive drugs, the virus can reactivate with 90

debilitating consequences [1,2]. Further, HCMV is the leading congenital infection in 91

the World, infecting up to 2.5% of live births and causing life-long neurological 92

defects [3]. 93

94

HCMV employs a range of immune evasion strategies to facilitate persistence. These 95

include down-regulation of MHC and co-stimulatory ligands and modulation of host 96

cytokine production [4,5]. Notably, HCMV encodes its own ortholog of the immune-97

regulatory cytokine interleukin (IL)-10 [6]. Two isoforms of viral IL-10 exist, both of 98

which suppress immune cell activation in vitro [7,8] and induce expression of cellular 99

IL-10 [9,10], suggesting the importance of the immune suppressive functions of IL-10 100

in HCMV infection in vivo. The murine CMV (MCMV) model is well established as a 101

model for HCMV infection in vivo due to similar cellular and tissue tropism, and 102

comparable anti-viral immune responses [11]. While MCMV does not encode a vIL-103

10, cellular IL-10 is induced upon infection of macrophages in vitro [12]. Data 104

obtained from the MCMV model has demonstrated an important protective role for 105

cellular IL-10 during acute CMV infection. Myeloid cells and B cells are the 106

predominant sources of IL-10 during initial MCMV infection [13,14]. IL-10 limits virus 107

induced weight loss, pro-inflammatory cytokine production and activation-induced NK 108

cell death [13-15]. Sustained acute MCMV replication in situations of high virus load 109

also induces IL-10 production by NK cells that restricts CD8+ T cell-mediated immune 110

pathology [16]. In contrast, production of IL-10 during chronic MCMV infection 111

5

suppresses viral clearance. Indeed, IL-10-deficient mice exhibit dramatic expansions 112

of virus-specific T cell responses, reduced virus persistence in the salivary glands 113

[14] and fewer viral genome copies in peripheral tissues during chronic/latent 114

infection [17]. Furthermore, persistent MCMV replication in the salivary glands is 115

dramatically restricted by the blockade of IL-10R signaling [18]. These data suggest 116

that although blocking the action of IL-10 during acute CMV infection may be harmful 117

to the host, targeting IL-10-mediated regulation of antiviral T cell responses may 118

impinge on virus chronicity and restrict horizontal virus transmission via mucosal 119

surfaces. 120

121

Previous studies have shown that TH1 cells can produce IL-10 under certain 122

conditions [19-24]. IL-10 producing TH1 cells have been shown to be protective in 123

parasitic infections by limiting infection-related pathology [24-26]. However, in the 124

context of lymphocytic choriomeningitis (LCMV) infection, virus replication is 125

accompanied by the production of IL-10+ T cells [27,28], and genetic deletion of IL-10 126

within T cells (or LysM+ cells) reduces virus chronicity [29]. HCMV-encoded latency 127

associated antigens induce CD4+/IL-10+ responses in healthy donors [30]. MCMV-128

specific IL-10 production by CD4+ T cells has also been described [17,31,32], and 129

CD4+ cell-derived IL-10 suppresses control of virus replication and leukocyte 130

accumulation during acute MCMV infection [33]. A substantial proportion of CD4+ T 131

cells express IL-10 upon polyclonal stimulation in salivary glands during MCMV 132

persistence [18]. However it is currently unknown how the expansion of these cells is 133

controlled during infection, and whether IL-10 production by T cells impacts on 134

MCMV chronicity . 135

136

Type-I IFNs are prototypic antiviral cytokines that exert important control of viral 137

replication. However inappropriate type-I IFN responses promote pathogenesis 138

associated with acute viral infections, and prolonged expression of type-I IFNs in 139

6

chronic viral infections including human immunodeficiency virus and hepatitis B virus 140

is implicated in driving immune pathology and antagonizing antiviral T cell responses 141

(reviewed in [34]). Further, in the LCMV model of infection, blockade of type-I IFN 142

receptor signaling in certain situations may enhance CD4+ T cell immunity and 143

control of virus chronicity, an outcome associated with reduced development of 144

immune-suppressive DCs [35,36]. Although type-I IFN is critical for early control of 145

CMV replication [37], the role that type-I has in shaping adaptive immunity during 146

CMV infection in incompletely understood. 147

148

The IL-12 family member IL-27 exhibits a broad spectrum of functions, including the 149

regulation of infection-induced pathologies (reviewered in [38]). Indeed, experiments 150

in murine influenza infection demonstrated that IL-27 restricts virus-induced 151

pathology associated with exuberant neutrophil, TH1 and TH17 responses [39]. IL-27 152

induces IL-10 expression by CD8+ T cells [40-42], and IL-27-dependent control of 153

influenza-induced inflammation is partially dependent upon IL-10 [39]. However, IL-154

27 impairs control of acute mouse hepatitis replication and associated pathology 155

following infection of the CNS and this phenotype is associated with reduced 156

accumulation of IL-10+ CD4+ T cells [43]. Paradoxically, IL-27 exerts cell intrinsic 157

positive regulation of antiviral CD4+ T cell responses during chronic LCMV infection, 158

promoting control of virus replication [44]. Moreover, IL-27 inhibits replication of both 159

HIV and HCV via the induction of interferon-stimulated genes [45,46], suggesting that 160

IL-27 is beneficial to the host during chronic viral infections. 161

162

Herein we investigated the role that IL-10+CD4+ T cells play in regulation of MCMV 163

specific T cell immunity. We identified that T cell-derived IL-10 suppressed T cell 164

response during persistent infection and subsequently promoted chronic virus 165

replication. We revealed that an axis involving type-I IFN and IL-27 promotes the 166

generation of peripheral IL-10-producing T cells that suppress antiviral immunity, 167

7

suggesting that CMV exploits this immune-regulatory pathway induced early in 168

infection to ensure viral persistence within mucosal tissue. 169

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Results 196

197

Virus-specific CD4+ T cells produce IL-10 during MCMV infection in peripheral 198

and mucosal tissue 199

We previously demonstrated that CD4+ but not CD8+ T cells produce IL-10 during 200

MCMV infection of the salivary glands, and that IL-10 promotes MCMV persistence 201

[18]. Therefore we sought to assess virus-specific CD4+ T cell production of IL-10 202

over time. Using a panel of MHC class II-restricted peptides identified to induce CD4 203

T cell responses during acute and persistent MCMV infection [32], we first defined 204

the antigen hierarchy of CD4+/IL-10+ production in both the mucosa and periphery 205

during MCMV infection using CD4-Cre-IL-10flox/flox (Cre-) mice, and mice lacking T cell 206

derived IL-10 (CD4-Cre+IL-10flox/flox, Cre+) as controls for IL-10 staining. In accordance 207

with data derived from polyclonally-stimulated CD4+ T cells [18], high frequencies of 208

virus-specific IL-10+CD4+ T cells were observed in salivary glands after MCMV 209

infection (Fig. 1A&B), peaking at d14 post-infection (pi). Frequencies of salivary 210

gland IL-10+CD4+ T cells were substantially higher than CD4+ T cells producing IFNγ 211

(Fig. 1C) suggesting that, at this time-point, IL-10+ T cells were the dominant MCMV-212

specific T cell response. We also detected virus-specific CD4+/IL-10+ T cells in the 213

periphery (Fig. 1A&D), highlighting that IL-10 production was not restricted to 214

mucosal CD4+ T cells [14,32]. The peripheral IL-10+CD4+ T cell response reactive to 215

M25, m142 and, to a lesser extent, m139, peaked during acute infection d7 pi (Fig. 216

1D) although, unlike salivary gland responses, were not present at higher 217

frequencies than IFNγ+ cells (Fig. 1E). Splenic IL-10+ CD4+ T cell frequencies also 218

contracted rapidly, remaining at low but significant levels during the remaining course 219

of infection. 220

221

222

223

9

Figure 1. Virus-specific CD4+ T-cells produce IL-10 during MCMV infection 224

CD4-Cre-IL-10flox/flox (Cre-) mice were infected with MCMV and at day 7, 14, 30 and 60 pi 225

leukocytes from the salivary glands (A-C &F) and spleen (A, &D-F) were isolated. (A) 226

Representative bivariant FACS plots of IL-10 versus IFNγ expression by viable (aqua 227

live/dead-) salivary gland (top) and splenic (bottom) CD4+CD3+ cells. CD4

+/IFNγ

+ and 228

CD4+/IL-10+ responses were measured after 6-hour stimulation with m09, M25, m139 229

and m142 MHCII MCMV antigens. Data is shown as mean ± SEM percent IL-10+ (B&D) 230

and percent + SEM IFNγ+ (C&E) CD4+ cells and (F) IL-10+, IFNγ

+ and double positive 231

cells over time. Data represents 8-16 mice per group and is representative of 2-3 232

experiments per time-point. 233

234

235

MCMV-specific CD4+/IL-10+ cells express T-Bet 236

MCMV-induced IL-10+CD4+ T cells do not express TH2-associated cytokines or the 237

regulatory T cell-associated transcription factor FoxP3 [18]. IL-10 is expressed by 238

numerous T helper subsets, including TH1 cells [24,25]. Interestingly, although a 239

small CD4+IFNγ+IL-10+ response was detected in the spleen after stimulation with 240

peptide for 6 hours, few co-producing cells were detected in the salivary glands upon 241

stimulation with cognate peptide (Fig. 1A&F), whereas MCMV-induced IL-10+CD4+ T 242

cells in the spleen and salivary glands can co-express IFNγ when poly-clonally 243

stimulated with PMA and ionomycin. 244

245

To further investigate the phenotype of IL-10+CD4+ T cells, we performed 246

transcription factor profiling of these cells using 10-BiT reporter mice that express the 247

surface marker CD90.1 (Thy1.1) under control of the IL-10 promoter without 248

impacting on endogenous IL-10 production [47]. We studied CD4+IL-10+ cells in the 249

spleen and salivary glands at peak responsiveness (d7 and d14, respectively). In 250

accordance with the ability to express IFNγ in response to polyclonal stimulation, 251

10

significant expression of the TH1-associated transcription factor T-Bet was observed 252

in salivary gland and, to a lesser extent, splenic Thy1.1+ CD4+ T cells (Fig. 2A-C). In 253

contrast, Thy1.1+ CD4+ T cells did not express the TH17-associated transcription 254

factor, aryl hydrocarbon receptor, nor did they co-express Tr1 cell markers CD49b 255

and LAG-3 (S1 Figure). Instead, splenic Thy1.1+ CD4+ T cells co-expressed the TH1-256

associated chemokine receptors CXCR3 and CCR5 (Fig. 2D). Although Thy1.1+ and 257

Thy1.1- salivary gland CD4+ T cells expressed low levels of CXCR3 and CCR5 14 258

days pi (Fig. 2D), high expression of their cognate chemokines during salivary gland 259

MCMV infection [48] is consistent with the hypothesis that these receptors are 260

internalized upon binding to their chemokine ligands within infected tissue. 261

262

To further explore the transcription control of IL-10 production by CD4+IL-10+ cells, 263

we examined the expression of Blimp-1 and c-Maf [49]. Significant expression of both 264

transcription factors was detected in Thy.1.1+ cells from the spleen and salivary 265

glands of infected mice (Fig. 2A-C). Furthermore, with the exception of Blimp-1 266

expression by salivary gland CD4+ T cells, increased expression of T-Bet, Blimp-1 267

and c-Maf by Thy1.1+ was measured as compared to Thy1.1- cells (Fig. 2A-C). 268

Overall, the combination of transcription factor and chemokine receptor expression of 269

IL-10+CD4+ T cells induced in response to MCMV indicated that these cells 270

originated from a TH1 lineage. 271

272

Figure 2. Transcription factor profiling of CD4+/IL-10+ producing T cells 273

10-BiT reporter mice were infected with MCMV and at day 7 and d14 pi spleen and 274

salivary glands were isolated. (A) Representative bivariant FACS plot of viable (aqua 275

live/dead-) Thy1.1 expression by CD4+CD3+ cells (top) and representative histograms of 276

T-Bet, BLIMP-1 and c-maf expression or (D) contour FACS plot of CXCR3/CCR5 277

expression by Thy1.1- or Thy1.1

+ CD4

+CD3

+ cells. Control = fluorescent minus one-278

stained Thy1.1+ samples from each time-point. (B&C) Intensity of transcription factor 279

11

staining by CD4+CD3+ cells in the spleen (B) and salivary glands (C) is shown as mean + 280

SEM of 5-6 mice/group and is representative of 2 separate experiments. 281

282

283

HCMV specific T-cells in peripheral blood and mucosal tissue secrete IL-10 284

We next asked whether HCMV-specific T cells reactive to common HCMV antigens 285

would also produce IL-10. Healthy colon and peripheral blood was obtained from 286

colorectal cancer (CRC) patients undergoing colonic resection and we assessed 287

glycoprotein B (gB, UL55) and tegument (pp65, UL83) specific IL-10/IFNγ production 288

using fluorospot. As compared to the low baseline IL-10/IFNγ production by medium-289

stimulated T cells (Fig. 3A), notable albeit variable frequencies of pp65 and gB 290

specific cytokine production were measured in blood (Fig. 3B). Interestingly, IL-10 291

production dominated pp65- and gB-specific responses in 3 of 4 and 2 of 4 patients, 292

respectively (Fig. 3B). Importantly, we also detected HCMV-specific cytokine 293

production in leukocytes isolated from the colon (Fig. 3A&B), demonstrating the 294

presence of HCMV-specific IL-10-producing T cells in human mucosal tissues. To 295

confirm HCMV-specific IL-10 production was derived from CD4+ T cells, larger 296

volumes (50mls) of blood were drawn from healthy donors, enabling the purification 297

of CD4+ T cells prior to peptide stimulation. Again, both CD4+/IFNγ+ and CD4+/IL-10+ 298

cells were observed following stimulation with pp65 and, to a far lesser extent, gB 299

peptide pools (Fig. 3A&B). As observed in MCMV infection (Fig. 1A&F), very few 300

virus-specific cells co-produced IL-10 and IFNγ (Fig. 3A&B). Thus, overall, these data 301

demonstrate the presence of HCMV-specific IL-10+ T cells in mucosal tissue and 302

peripheral blood. 303

304

Figure 3. HCMV specific T-cells in peripheral blood and mucosal tissue secrete 305

IL-10 306

(A) Total leukocytes from colon and peripheral blood (left) or peripheral blood CD4+ T-307

12

cells isolated by negative selection (right) were isolated from colorectal patients 308

undergoing surgery (left) or HCMV sero-positive healthy donors (right) were stimulated 309

with γ-irradiated autologous targets that were pre-pulsed with vehicle control, pp65 or gB 310

peptide pool. γ-irradiated autologous targets pre-pulsed with vehicle control or pp65 is 311

also shown from healthy CMV sero-positive individuals. After 16hrs, cytokine expression 312

was analyzed using a human IFNγ/IL-10 fluorospot kit where green spots represent 313

IFNγ+ cells, red spots represent IL-10+ cells and yellow spots indicate dual IFNγ

+/IL-10+ 314

cells. (B) Peptide-specific spot forming cell (SFC) were calculated after subtraction of 315

SFCs in medium-stimulated wells. Data is expressed as mean + SEM of duplicate 316

values. Individual donors are shown. 317

318

319

T cell derived IL-10 restricts MCMV-specific T cell immunity 320

We sought to investigate the impact of IL-10 production by CD4+ T cells on CMV-321

specific immunity in vivo. We utilized CD4-Cre+IL-10flox/flox (Cre+) mice in which CD4+ 322

cells do not produce IL-10. We detected no compensatory IL-10 production by other 323

leukocytes in response to MCMV in these mice (S2 Figure). As compared to CD4-324

Cre-IL-10flox/flox (Cre-) control mice, Cre+ mice mounted elevated CD4+/IFNγ+ 325

responses in the salivary glands to multiple MCMV antigens throughout the course of 326

infection (Fig. 4A), most notably M25- and m142-specific cells at d14 pi when peak 327

IL-10+ responses were observed in Cre- mice (Fig. 1B). The absence of CD4+ cell-328

derived IL-10 also consistently increased the accumulation of peripheral virus-329

specific IFNγ+ CD4+ T cells during the persistent phase of infection from d14 pi (Fig. 330

4B), supporting the hypothesis that initial production of IL-10 by T cells in the 331

periphery impinged on later TH1 cell accumulation. 332

333

It has been shown that IL-10 deficiency leads to an increased splenic DC 334

accumulation and expression of co-stimulatory ligands during acute infection [14]. 335

13

Cre+ mice exhibited no notable increased accumulation of splenic DCs or salivary 336

gland myeloid cells either d7 or d14 pi (S3 Figure). However, we observed an 337

increased frequency of myeloid cells expressing co-stimulatory ligands previously 338

demonstrated to enhance MCMV-specific T cell responses during virus persistence 339

[50-52], in the spleens (d7 and d14 pi) and salivary glands (d7 pi) of Cre+ mice (S3 340

Figure). Although we report some inter-experiment variability regarding the impact of 341

T cell-derived IL-10 on myeloid cell CD86 expression, collectively these data are 342

consistent with a role for T cell-derived IL-10 in modulating myeloid cell function. 343

Furthermore, in accordance with elevated CD4+ T cell responses in Cre+ mice (Fig. 344

4A&B), we observed an increased accumulation of MCMV-specific CD8+ T-cells in 345

peripheral tissues (Fig. 4C). As observed in studies of IL-10-/- mice [17], T cell-346

derived IL-10 preferentially suppressed CD8+ T cell responses reactive to IE3 (Fig. 347

4C), consistent with observation that IE3-specific CD8+ T cell inflation is particularly 348

dependent upon CD4+ T cell help [53]. 349

350

Figure 4. T cell-derived IL-10 impairs anti-MCMV T cell immunity and promotes 351

virus persistence 352

CD4-Cre-IL-10flox/flox(Cre-) and CD4-Cre+IL-10flox/flox (Cre+) mice were infected with MCMV 353

and at day 7, 14, 30 and 60 pi peptide-specific CD4+ IFNγ

+ responses in the salivary 354

glands (A) and spleen (B) were measured. Data is shown as mean ± SEM of cell 355

numbers with mean of 8-17 mice per group. (C) Splenic (top) and pulmonary (bottom) 356

virus-specific CD8+ T cells were quantified with tetramers refolded around peptides from 357

MCMV antigens IE3, M38, m139 and M45. Total CD8+/CD3+ Tetramer+ cells are plotted 358

with 4-8 mice per group representative of 2 experiments. (D) Replicating virus in salivary 359

gland homogenates at day 7, 14 and 28 pi were measured by plaque assay. Data is 360

shown as individual mice + median and represents 2-3 experiments. (E) Viral genomes 361

were measured in saliva by qPCR day 7, 14, 21 and 28 pi. Data is shown as mean ± 362

SEM from 5 mice/group from 3 replicative experiments. (F) Replicating virus in salivary 363

14

gland homogenates d14 pi in rag1-/-

mice following transfer of WT or IL-10-/- CD4

+ T cells. 364

Data is shown as individual mice + median and represents two separate experiments. 365

366

367

The absence of IL-10 production by CD4+ T cells controls viral replication 368

without inducing autoimmunity 369

Unlike experiments performed in BALB/c mice [33], we detect no significant virus 370

replication in the salivary glands during acute infection of C57BL/6 mice (Fig. 4D). 371

Reduced persistent virus replication is observed in salivary glands of Il-10-/- mice [14] 372

and in WT C57BL/6 mice in which IL-10R signaling is antagonized [18]. Associated 373

with elevated CD4+ T cell responses d14 pi in Cre+ mice, fewer replicating virions 374

were detectable in salivary glands as compared to controls. Furthermore, 5 out of 10 375

Cre+ mice had cleared replicating MCMV by d30 pi whereas MCMV replication was 376

detectable in all Cre- mice (Fig. 4D). Importantly, reduced virus load in Cre+ mice was 377

accompanied by decreased shedding of virus in the saliva throughout the course of 378

infection (Fig. 4E). Furthermore, improved control of virus replication in Cre+ mice 379

was not accompanied by virus-induced Sjögrens Syndrome-like disease, as 380

demonstrated by comparable salivary gland accumulation of TRAILR-expressing 381

CD4+ T cells and serum Sjögren Syndrome Antigen (SSA)-specific IgG (S4 Figure), 382

both of which are implicated in this disease [54]. 383

384

One possible caveat of studies using CD4-cre mice is that CD8+ T cells (that express 385

CD4 during thymic development) and CD4+ dendritic cells also lack IL-10 expression. 386

To prove that IL-10 derived from CD4+ T-cells facilitated MCMV persistence, we 387

negatively selected CD4+ T-cell from WT or Il-10-/- mice and adoptively transferred 388

cells into rag1-/- mice. Whereas transfer of WT CD4+ T cells reduced MCMV titers by 389

1 log 14d pi, Il-10-/- T cells reduced virus load by 2 logs (Fig. 4F), comparable to virus 390

15

load in Cre+ mice at this time-point (Fig. 4D). Thus, two independent approaches 391

supported the conclusion that CD4+ T cell derived IL-10 facilitate MCMV persistence. 392

393

MCMV-induced IL-27 is secreted during acute infection and promotes the 394

development of splenic IL-10+ CD4+ T cells 395

We sought to define the factors promoting the generation of CD4+IL-10+ T cells. IL-27 396

is a potent inducer of IL-10 production [38]. We detected a substantial IL-27 p28 397

production during acute MCMV infection in the spleen that peaked d2 pi (Fig. 5A). 398

Elevated IL-27 production in the salivary glands during viral persistence was also 399

detected, albeit much lower than concentrations measured in the spleen (Fig. 5A). IL-400

27 is produced by multiple cells types with predominant expression by myeloid cells 401

[55]. In accordance, we detected a high frequency of IL-27+ splenic DCs (Fig. 5B&C) 402

and, to a lesser extent, macrophages, neutrophils and also B cells (Fig. 5B&C). 403

404

Figure 5. MCMV infection induces myeloid cell expression of IL-27 during acute 405

infection 406

Spleen and salivary glands were isolated from naïve Cre- mice or after 2 and 4 days of 407

infection. (A) IL-27 release was measured in tissue homogenate supernatants by ELISA. 408

(B&C) IL-27p28 expression by DCs (MHCII+/CD11c+/Ly6C+), Macrophages 409

(CD11b+/F480+/Ly6C+), B-cells (B220+/Siglec-H-), Neutrophils (Ly6G+) and T-cells (CD3+) 410

was assessed by intracellular cytokine staining and is shown as mean + SEM of %IL-27 411

release or by representative concatenated bivariate FACS plots (C). Gating was 412

determined using fluorescent minus one-stained samples from d2-infected mice. Data is 413

shown as 8 mice per group and is representative of at least 2 experiments. 414

415

416

To establish the link between CMV-induced IL-27 production, and the generation of 417

IL10+CD4+ T cells, the following experiments were conducted. Il-27rα-/- (wsx1-/-) mice 418

16

were employed which do not respond to IL-27 as a consequence of the loss of the IL-419

27 receptor complex. Development of splenic IL-10+CD4+ T cells during acute 420

infection was almost completely abrogated in Il-27rα-/- (wsx1-/-) mice, as were the 421

smaller frequencies of IL-10+ cells present d14 pi (Fig. 6A&B). In contrast, 422

development of IL-10+CD4+ T cells in the salivary glands was comparable in WT and 423

Il-27rα-/- mice (Fig. 6A). The common IL-27 cytokine receptor glycoprotein 130 424

(gp130) is down-regulated by virus-specific memory T cells [41] and IL-10+ (Thy1.1+) 425

and IL-10- (Thy1.1-) CD4+ T cells in the salivary glands d14 pi expressed less gp130 426

than splenic T cells isolated d7 pi (Fig. 6C&D). Importantly, salivary gland IL-10+ T 427

cells expressed high levels of the co-stimulatory molecule ICOS (Fig. 6E) that has 428

previously been implicated in the development of IL-10-secreting CD4+ T cells during 429

viral infection [56]. Monoclonal antibody blockade of ICOS from 6 days pi significantly 430

inhibited the development of salivary gland MCMV-specific IL-10+CD4+ T cells 14 431

days pi (Fig. 6F). In contrast, splenic IL-10+CD4+ T cells were unaffected by ICOS 432

blockade (Fig. 6G). These data suggest that differential signals are responsible for 433

induction of MCMV-specific IL-10+ T cells in different tissues during MCMV infection. 434

435

Figure 6. IL-27 promotes splenic CD4+IL-10+ T cell development whereas ICOS is 436

required for salivary gland CD4+IL-10+ T cell accumulation. 437

Il-27rα-/- or WT (C57BL/6) mice were infected with MCMV and spleen and salivary 438

gland CD4+/IL-10+ (A) responses were quantified and expressed at mean + SEM of 439

11 mice/group. (B) Representative bivariant FACS plots of IFNγ versus IL-10 440

expression by splenic CD4+CD3+ T cells. (C&D) gp130 and (E) ICOS expression by 441

IL-10+ (Thy1.1+) and IL-10- (Thy1.1-) CD4+CD3+ T cells was assessed in 10-Bit mice 442

and shown as representative FACS plots (C) and histogram overlays (E) with mean + 443

SEM of 5-6 mice/group (D). Gating was determined using Thy1.1- CD4+CD3+ cells 444

derived from fluorescent minus one-stained samples from mice infected for 14 days. 445

(F&G) WT (C57BL/6) mice were infected with MCMV and at d6 and d10 pi αICOS or 446

17

Isotype antibody was added. At d14 pi (A) salivary glands and (B) spleen CD4+/IL-447

10+ responses were quantified and expressed at mean + SEM of 6 mice/group. 448

449

450

IL-27 suppresses the development of antiviral TH1 immunity and promotes 451

MCMV persistence 452

Given the reduced splenic IL-10+CD4+ T cell accumulation in Il-27rα-/- mice, we asked 453

whether antiviral TH1 responses were enhanced. IL-27R deficiency did not influence 454

virus-specific TH1 responses during acute infection (Fig. 7A). However, in 455

accordance with data derived from Cre+ mice, Il-27rα-/- mice exhibited a significantly 456

increased accumulation of virus-specific TH1 cells within the periphery and salivary 457

glands during the persistence phase of infection (Fig. 7A). As observed in Cre+ mice 458

(Fig. 4C), elevated TH1 cell were also accompanied by increased virus-specific CD8+ 459

T-cell response d30 pi (S5 Figure). In contrast, blockade of ICOS from 6 days pi did 460

not enhance virus-specific IFNγ+CD4+ T cell responses either in the spleen or 461

salivary glands 14 days pi (S6 Figure), suggesting a dominant inhibitory effect of IL-462

27-dependent IL-10+ T cell responses during the first 14 days of MCMV infection. 463

464

Given that IL-27 induction of IL-10 significantly impaired anti-MCMV T cell immunity, 465

our attention focused on whether IL-27 impacted on viral persistence. In the absence 466

of IL-27 signaling, viral load was decreased d14 pi and MCMV replication was absent 467

in salivary glands of most mice by d30 pi (Fig. 7B). Furthermore, we detected a 468

significant decrease in virus shedding by Il-27rα-/- mice in the saliva, with few viral 469

genomes detected in the saliva by d30 pi (Fig. 7C). These data are consistent with 470

the hypothesis that IL-27 induction of IL-10+CD4+ T cells in the periphery during 471

acute infection contributes to the suppression of TH1 immunity and subsequent viral 472

persistence and transmission. 473

18

Figure 7. IL-27 promotes CD4+IL-10+ development and impairs anti-MCMV TH1 474

immunity and control of MCMV persistence 475

Il-27rα-/- or WT (C57BL/6) mice were infected with MCMV and spleen and salivary 476

gland CD4+/IFNγ+ (A) responses were quantified and expressed at mean + SEM of 477

11 mice/group. (B) Replicating virus in salivary gland homogenates of Il-27rα-/- and 478

WT mice is shown as individual mice + median. Data is representative of 2 479

experiments. (C) MCMV genomes in saliva were quantified by qPCR. Data is shown 480

as mean ± SEM from 11 mice per group from 2 replicative experiments. 481

482

483

MCMV-induced IL-27 production during acute infection is dependent upon 484

type-I IFN and promotes virus persistence 485

We examined the factors regulating IL-27 production in response to MCMV. Type-I 486

interferon exerts critical control of MCMV infection in vivo [57,58]. However, type-I 487

IFN induction of IL-27 during bacterial infection has been described [59]. Hence a 488

paradoxical situation may arise where type-I IFN controls CMV on one hand, but 489

induces IL-27 leading to inferior control of persistent virus replication. We examined 490

whether type-I IFN orchestrated IL-27 production in response to MCMV, using an 491

established in vitro MCMV infection system of bone marrow-derived macrophages 492

[60]. Productive infection of WT macrophages induced substantial IL-27 mRNA 493

expression, peaking 6hrs pi (Fig. 8A). Similarly, IL-27 expression was also maximally 494

induced 6hrs pi with by a non-productive viral replication, as demonstrated using 495

replication-deficient (ΔIE3) MCMV. As the primary macrophage response to viral 496

infection involves type-I IFN signaling we next directly tested the dependency of IL-27 497

induced expression in macrophages that are genetically ablated for either IFNβ 498

production or type-I IFN signaling. MCMV infection of either Ifnβ-/- or Ifnαr-/- 499

macrophages failed to induce IL-27, a result further mirrored by IFN-dependent IL-27 500

expression upon stimulation with the TLR3 agonist, Poly:IC (Fig. 8A). Moreover, 501

19

administration of a blocking anti-IFNαR-1 monoclonal antibody during MCMV 502

infection in vivo inhibited IL-27 expression by all cellular subsets examined (Fig. 8B). 503

In the case of macrophages, B-cells and neutrophils, IL-27 production was almost 504

completely abrogated by IFNαR-1 blockade (Fig. 8B). To examine whether early IFN-505

induced IL-27 production shaped antiviral responses, we treated MCMV-infected 506

mice with neutralizing anti-IL-27 antibody at the time of infection (Fig. 8C). IL-27 507

neutralization significantly enhanced total virus-specific TH1 responses and reduced 508

virus shedding 14 days pi (Fig. 8D&E) whilst concurrently reducing accumulation of 509

virus-specific IL-10+CD4+ T cells in the spleen at this time (Pooled m09, M25, m139 510

and m142-specific CD4+IL-10+ T cells: Isotype control = 0.00162 × 107 cells versus 511

αIL-27 treated group = 0.000908 × 107 cells). Importantly, IL-10R blockade from the 512

time of IL-27-induced IL-10+ T cell responses (from day 6 pi) had no additional 513

inhibitory influence on IFNγ+ T cell responses and virus shedding (Fig. 8D&E). Thus, 514

IL-27 production in response to MCMV is dependent upon type-1 IFNR signaling, and 515

this axis acts during the initial days of infection to promote the accumulation of 516

MCMV-specific IL-10+CD4+ T cells, subsequently promoting virus persistence and 517

shedding from the mucosa. 518

519

Figure 8. Type-I IFN induces IL-27 expression during MCMV infection 520

(A) Heat map of IL-27 p28 mRNA expression by WT, IFNβ-/-

and IFNαR-/-

macrophages 521

following stimulation with WT MCMV (C3X), replication-deficient (ΔIE3) MCMV or 522

poly(I:C). (B) Expression of IL-27 p28 by splenic leukocytes was assessed d2 pi in Cre- 523

mice treated/not with 2 mg anti-IFNαR-1. Mean + SEM of 8 mice/group is shown and 524

data is representative of 2 separate experiments. (C) WT (C57BL/6) mice were infected 525

with MCMV treated with anti-IL-27, Isotype or virus alone. At d6 pi mice were treated 526

/not with anti-IL-10R and at d14 pi spleen CD4+/IFNγ+ (D) responses were quantified 527

and expressed as mean ± SEM of 4-15 mice/group. (E) MCMV genomes in saliva 528

were quantified by qPCR. Data shown as mean ± SEM from 4-15 mice/ group. 529

20

Discussion 530

531

Herein we reveal that cytokine responses induced upon acute MCMV infection 532

influence antiviral T cell responses and virus replication during pathogen persistence. 533

We identified that IL-27, which was produced by myeloid cells during the initial days 534

of infection, promoted the generation of IL-10+CD4+ T cells in the spleen. Our data 535

suggest that this IL-27-dependent induction of peripheral IL-10+CD4+ T cells was 536

sufficient to impinge on T cell mediated control of virus chronicity in the mucosa. IL-537

27 production was dependent upon type I IFN, implying that CMV exploits the 538

immune-regulatory actions of this prototypic antiviral cytokine pathway to enable 539

persistence and dissemination. 540

541

The dynamics of virus-specific IFNγ versus IL-10 production by CD4+ T cells 542

suggested that contraction of the IL-10 response preempts expansion of IFNγ+ T cell 543

numbers and subsequent control of virus replication. Preventing IL-10 production by 544

T cells both enhanced and accelerated the generation of virus-specific TH1 545

responses. These data suggest that IL-10+ T cells provide a window of opportunity 546

for MCMV to replicate within and shed from the salivary glands. Conversely, these 547

results also imply that inhibiting virus-specific IL-10+ T cell development may be 548

therapeutically beneficial. Our data suggest that peripheral IL-10+ T cells inhibit the 549

control of virus replication within mucosal tissue independently of mucosal IL-10+ T 550

cell populations, highlighting that appropriate modulation of peripheral T cell 551

responses via systemic vaccination approaches may be capable of overcoming 552

inhibitory pathways that act in the mucosa, thus enabling effective control of virus 553

replication within mucosal tissues. 554

555

Using a combination of conditional knockout mice and a cell transfer model, we 556

demonstrated an important role for CD4+ T cell-derived IL-10 in facilitating MCMV 557

21

persistence. This result is in apparent contradiction to bone marrow chimera 558

experiments suggesting no role for CD4+ cells in IL-10-mediated chronic MCMV 559

replication [14]. There are several explanations for these apparent discrepancies. 560

Firstly, whereas we used salivary gland-propagated Smith strain MCMV in our 561

experiments, Mandaric et al examined tissue culture-propagated virus deficient in 562

m157, a viral ligand for the NK cell activating receptor Ly49H [61]. These viruses 563

likely replicate with different kinetics and, possibly, tropisms that may influence the 564

induction of IL-10 expression by T cells and other cells. Furthermore, the mixed 565

chimera experiments performed by Mandaric et al used IL-10-/- mice as recipients 566

whereas our experiments were performed in mice from IL-10-sufficient backgrounds. 567

Given that the time-point assessed by Mandaric et al (d14 pi) was prior to the 568

maximal impact of CD4-derived IL-10 in our model, such variations in experimental 569

design may influence the impact of CD4+ T cell-derived IL-10 on virus persistence. 570

571

Importantly however, significant expression of IL-10 by HCMV-specific T cells in 572

peripheral blood and mucosal tissue highlights the potential importance of T cell-573

derived IL-10 in regulating anti-CMV immunity and viral persistence, as suggested by 574

findings obtained from our model of chronic MCMV infection, and further supported 575

by experiments studying acute MCMV infection [33]. HCMV-specific IL-10 production 576

was detected in response to common lytic antigens pp65 and gB, although T cell 577

responsiveness to gB was low in peripheral blood of all healthy volunteers examined. 578

IL-10 production by CD4+ T cells reactive to latency-associated HCMV antigens have 579

also been described [30]. Persistent shedding of HCMV shedding in children is 580

associated with poor TH1 responses [62]. Our data derived from murine experiments 581

demonstrates that preventing the generation of HCMV-specific IL-10+ CD4+ T cells 582

may be beneficial for the improvement of protective T cell immunity following 583

vaccination, particularly in the context of restricting horizontal transmission via 584

mucosal surfaces. 585

22

IL-27 promoted the development of splenic but not salivary gland IL-10+CD4+ T cells. 586

Why IL-27 had no impact on salivary gland CD4+IL-10+ accumulation is incompletely 587

understood. Salivary glands APCs, which are phenotypically indicative of tissue-588

resident macrophages [63], are tightly regulated via inhibitory signals such as CD200 589

expressed by endothelial cells [60]. Thus, restricted activation of this cell population 590

may contribute to the relatively low IL-27 production measured in this organ. 591

Furthermore, the infection phase may also influence the impact of IL-27 on IL-10+ T 592

cell development. Memory CD8+ T cells generated in influenza infection down-593

regulate gp130 [41] and CD4+ T cells in persistently-infected salivary glands 594

expressed less gp130 expression than splenic CD4+ T cells during acute infection. 595

This down-regulation of gp130 correlated with a reduced impact of IL-27R signaling 596

on salivary gland IL-10+ CD4+ T cell accumulation during virus persistence. 597

598

Instead, ICOS co-stimulation was required for the accumulation of IL-10+CD4+ T cells 599

within the salivary glands, thus demonstrating that differential signals promote IL-10+ 600

T cell responses in different anatomical locations of MCMV infection and suggesting 601

that ICOS acts independently of IL-27 in the induction of salivary gland CD4+IL-10+ T 602

cells. This contrasts in vitro findings demonstrating that ICOS acts downstream of IL-603

27 in promoting IL-10 production by Tr1 cells in vitro [64]. However, salivary gland IL-604

10+CD4+ T cells in our model were phenotypically distinct from Tr1 cells. Moreover, 605

here we examined the influence of ICOS co-stimulation of T cells during virus 606

persistence rather than during initial T cell activation as studied by Pot et al. 607

Therefore, although the impact of ICOS on CD4+IL-10+ T cell generation during acute 608

infection was not examined in our study, these data collectively imply that the type of 609

IL-10-producing T cell and the timing of IL-10-inducing signals may influence the 610

relationship between IL-27 and ICOS in the induction of CD4+IL-10+ T cells. Notably, 611

blockade of ICOS was not accompanied by an elevated virus-specific TH1 responses 612

d14 pi despite dramatically reducing salivary gland CD4+IL-10+ accumulation. This 613

23

may reflect a possible dual function for ICOS in positive co-stimulation [65] in addition 614

to IL-10 induction. However, our data does not preclude a possible role for ICOS and, 615

possibly, other IL-10-inducing receptors in the suppression of antiviral control during 616

the latter stages of MCMV persistence. 617

618

We demonstrate that IL-27 is critical for the induction of peripheral IL-10+CD4+ T cell 619

responses and subsequent suppression of TH1 responses during the persistent 620

phase of infection. Experiments using anti-IL-27 and anti-IL-10R suggest that early 621

IL-27-mediated induction of IL-10+ T cells has a long-term impact on antiviral 622

immunity and virus persistence. However, our data does not preclude an IL-10-623

independent inhibitory function for IL-27 during the later stages of infection. Indeed, 624

antagonizing both IL-10R and IL-27 14 days pi led to an additive, albeit modest, 625

increase in virus-specific IFNγ+CD4+ T cells by day 30 (S7 Figure). Intriguingly, IL-27 626

can induce dendritic cell expression of the ATPase CD39 [66]. However, in contrast 627

to IL-27rα-/- mice, mice deficient in the ecto-5’-nucleotidase CD73 that acts 628

downstream of CD39 in the purinergic system have no alteration in MCMV-specific 629

memory T cell accumulation and only a moderate and transient reduction in MCMV 630

replication in the salivary glands [67]. Taken together with data derived from 631

CD4creIL-10flox and IL-27rα-/- mice, these results collectively suggest that early 632

induction of IL-10+CD4+ T cells is the dominant mechanism through which IL-27 633

impinges on anti-MCMV T cell immunity. 634

635

In vivo and in vitro experiments revealed that the induction of IL-27 during MCMV 636

infection was promoted by type-I IFN. Although the uncontrolled virus replication and 637

associated disease and inflammation observed in mice treated with anti-IFNαR-1 638

antibody precluded the assessment of the direct impact of IFNαR-1 signaling on both 639

IL-27-driven IL-10+ T cell development and virus persistence, our data implies that 640

type-I IFN may impinge on antiviral immunity via the induction of IL-27. Comparable 641

24

IFN-dependent induction of IL-27 was observed upon in vitro stimulation with either 642

MCMV or the TLR3 ligand, poly(I:C). Endosomal TLRs including TLR3 are activated 643

upon acute MCMV infection and induce type-I IFN expression [68,69]. Thus, our data 644

suggest that MCMV may exploit an immune-regulatory aspect of this “antiviral” 645

response to promote persistence within mucosal tissue and subsequently increase 646

the window of opportunity for horizontal transmission via mucosal secretions. 647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

25

Materials and Methods 670

671

Mice, infections and treatments 672

CD4-Cre-IL-10flox/flox (Cre-)/CD4-Cre+IL-10flox/flox (Cre+) were generated by Werner 673

Muller (University of Manchester) and were kindly provided by Jean Langhorne 674

(Francis Crick Institute), 10-BiT mice were given to us by Padraic Fallon (Trinity 675

College Dublin) and Il-27rα-/- (wsx1-/-), Rag1-/- and Il-10-/- mice were obtained from 676

The Jackson Laboratory. All mice were bred in-house. C57BL/6 WT mice were 677

purchased from Charles River or Envigo. Mice were infected with MCMV that was 678

prepared by sorbital gradient purification as described previously [70]. Mice were 679

infected with 3 × 104 pfu MCMV intraperitoneally (i.p.). In some experiments, CD4-680

Cre-IL-10flox/flox mice were administered 2 mg αIFNαR-1 (clone MAR1-5A3, BioXcell) 681

or IgG1 Isotype control (clone MOPC-21 BioXcell) at the time of infection, or 200 µg 682

anti-ICOS (clone 7E.17G9 or JmAb51-TM) or IgG control (clone LTF2 or R347-TM), 683

respectively) on days 6 and 10 pi. In other experiments mice were treated with 500 684

µg anti-IL-27 (clone MM27-7B1, Biolegend), 250 µg anti-IL-10R (clone 1B1.3A 685

BioXcell) and also in combination or Isotype control (clone MOPC-21 BioXcell) on 686

days stated in the legend. In some experiments, CD4+ T cells were isolated from 687

spleens of C57BL/6 and IL-10-/- mice by negative separation (Miltenyi). 5 x 106 cells 688

were then transferred intravenously (i.v.) into rag1-/- mice one week prior to MCMV 689

infection. 690

691

T-cell effector function assays 692

Leukocytes were extracted from murine spleen, salivary glands and lungs as 693

described previously [13,60]. For CD4+ functional responses, isolated leukocytes 694

were stimulated for 2 hours with 3 µg/ml m09, (GYLYIYPSAGNSFDL), M25 695

(NHLYETPISATAMVI), m139 (TRPYRYPRVCDASLS), and m142 696

(RSRYLTAAAVTAVLQ) MCMV MHCII peptides (Genscript). Brefeldin A (Sigma) was 697

26

then added and cells incubated for a further 4 hours. For CD8+ functional responses, 698

leukocytes were stimulated with 2 µg/ml m139, IE3, M38, and M45 MCMV MHCI 699

peptides in the presence of anti-mouse CD107a-FITC (Biolegend) with monensin 700

(BD Biosciences) and brefeldin A for 6 hours. Cells were subsequently stained with 701

Zombie Aqua fixable viability dye (Biolegend) or LIVE/DEAD-Aqua (Life-702

Technologies), stained with anti-CD16/CD32 Fc-block (Biolegend) and then with 703

either anti-CD4 Pacific-Blue or PercP, (clone RM4-5, Biolegend) or with anti-CD8 704

PercP (clone 53-6.7, Biolegend) and anti-CD262 (TRAILR DR5, clone MD5.1, 705

eBioscience). Cells were fixed, saponin permeabilised and stained for anti-IFNγ FITC 706

or Pacific-Blue (clone XMG1.2, Biolegend) and anti-IL-10 APC (clone JES5-16E3, 707

eBioscience). Data was acquired using a BD FACSCantoII flow cytometer (BD 708

Biosciences) and analysed with FlowJo software (TreeStar). 709

710

Cell phenotyping and IL-27 detection 711

Direct ex vivo intracellular IL-27 production was detected as previously described 712

[71]. Cells were stained with a combination of anti-I-A/I-E (clone M5/114.15.2, 713

Biolegend), anti-CD11c Pe-Cy7 (clone HL3 BD, Pharmingen), anti-Ly6C FITC (clone 714

AL-21 BD Pharmingen), anti-CD11b APC-Cy7 (clone M1/70, Biolegend), anti-F480 715

Brilliant-Violet 711 (BV711) (clone BM8, Biolegend), anti-CD45R/B220 BV785 (clone 716

RA3-6B2, Biolegend), SiglecH APC (clone 551, Biolegend), anti-Ly6G PerCP-Cy5.5 717

(clone 1A8, Biolegend), and anti-CD3ε BV605 (clone 145-2C11, Biolegend). Cells 718

were fixed, saponin permeabilised and stained with anti-IL-27 p28 PE (clone MM27-719

7B1, Biolegend). To determine T cell transcription factor profiles cell were surface-720

stained with anti-CD4 BV785 (clone RM4-5, Biolegend), anti-CD90/CD90.1 (Thy1.1) 721

BV650 (clone OX-7, Biolegend) anti-CD130 PE (gp130) (clone KGP13, eBioscience), 722

fixed and permeabilised using BD Cytofix/Cytoperm solution (BD Biosciences) and 723

stained with anti-T-Bet BV421 (clone 4B10, Biolegend), anti-BLIMP-1 Alexa-Fluor 724

647 (AF647) (clone 5E7, Biolegend), and anti-cMaf eFluor 660 (clone sym0F1, 725

27

eBioscience). All data was acquired using a BD LSRForetssa flow cytometer (BD 726

Biosciences) and analyzed with FlowJo software (TreeStar). For analysis of co-727

stimulatory ligand expression, cells were surface stained with a combination of anti-728

CD11c APC-Cy7 (clone N418, Biolegend), anti-I-A/I-E PerCP-Cy5.5 (clone 729

M5/114.15.2, Biolegend) anti-F480 Brilliant-Violet 421 (BV421) (clone BM8, 730

Biolegend), anti-CD80 APC (clone 16-0A1, Biolegend), anti-CD86 Pe-Cy7 (clone GL-731

1, Biolegend), anti-OX40L APC (clone RM134L, Biolegend), anti-4-1BBL PE (clone 732

TKS-1, Biolegend), anti-CD40 FITC (clone 3/23, Biolegend) and anti-Ly6G PE (clone 733

1A8, Biolegend), anti-ICOS APC-Cy7 (clone C398.4A, Biolegend), anti-AhR Alexa-734

Flour 488 (AF488) (clone 4MEJJ eBioscience), anti-CD49b Pe-Cy7 (clone DX5, 735

Biolegend), anti-LAG3 PE (clone C9B7W, Biolegend) anti-NK-1.1 Pe-Cy7 (clone 736

PK136, Biolegend), anti-CD19 FITC (clone 6D5, Biolegend), anti-CXCR3 PerCP-737

Cy5.5 (clone CXCR3-173 Biolegend), and anti-CCR5 AF488 (clone HM-CCR5, 738

Biolegend). Data was acquired using a BD FACSCantoII flow cytometer (BD 739

Biosciences) and analysed with FlowJo software (TreeStar). 740

741

Tetramer staining 742

The following MCMV MHCI biotinylated were refolded and kindly provided by the NIH 743

tetramer Core Facility- H-2D(b) M45 985-993 HGIRNASFI, H-2k(b) m139 744

TVYGFCLL, H-2k(b) M38 316-323 SSPPMFRV, H-2k(b) IE3 RALEYKNL. 745

Biotinylated monomers were tetramerised as described previously [72]. Isolated 746

leukocytes were stained with viability dye and then with 25 µg/ml tetramer conjugated 747

to PE or APC for 15 minutes at 37°C. Cells were subsequently stained with Fc-block 748

and anti-CD8 APC-Cy7 (clone 53-6.7, Biolegend). Data was acquired using a BD 749

FACSCantoII flow cytometer (BD Biosciences) and analysed with FlowJo software 750

(TreeStar). 751

752

Isolation of human T cells from human colon 753

28

Peripheral blood and healthy colon specimens were obtained from patients 754

undergoing primary tumor resection for colorectal adenocarcinoma at the University 755

Hospital of Wales, Cardiff. Blood samples were collected on the day of but prior to 756

surgery. The tissue used in this study was autologous colon samples that were cut 757

from a macroscopically normal section of the excised tissue, at least 10 cm from the 758

tumor. Colon tissue was washed in extraction medium consisting of DMEM 759

supplemented with 100 U/ml Penicillin, 100 mg Streptomycin and 2 mM L-Glutamine, 760

20 µg/ml Gentamincin and 2 µg/ml Fungizone (all Life-Technologies) as described 761

previously [73]. Samples were finely cut with scalpel blades in a petri dish and forced 762

through a 70 µM and subsequently a 40 µM cell strainer. Cells were washed and 763

pelleted by multiple centrifugations and lymphocytes were isolated using 764

Lymphoprep (Axis-Shield, Scotland). 765

766

Fluorospot analysis of IFNγ and IL-10 767

Peripheral blood mononuclear cells (PBMCs) from healthy HCMV sero-positive 768

healthy volunteers were isolated using Lymphoprep. For target cells, some PBMCs 769

were γ-irradiated with 3000 Rad, plated out @ 1 × 105 cells/well and pulsed with 2 770

µg/ml of Peptivator CMV pp65 overlapping peptide pool (Miltenyi Biotec), 2 µg/ml 771

PepMix HCMVA UL55 overlapping peptide pool (JPT), or with DMSO vehicle control 772

for 2 hours @ 37°C. CD4+ T cells were then isolated from the remaining PBMCs by 773

negative selection (Miltenyi Biotec). Pulsed autologous targets were then transferred 774

to a human IFN+/IL-10+ Flurospot plate thus removing (IL-10-producing) monocytes 775

that adhered to the plate. 3 × 105 purified CD4+ T-cells and 1/100 human anti-776

CD28/CD49d co-stimulation antibodies (BD Pharmingen) were then added for 18 777

hours @ 37°C. E:T ratios ranging from 1:3 to 3:1 were tested. After incubation, plates 778

were assayed according to the manufacturers instructions (Mabtech) and quantified 779

using a CTL Immunospot Fluorospot Line plate reader (CTL). Cells were maintained 780

in RPMI, 100 U/ml Penicillin, 100 mg Streptomycin, 2 mM L-Glutamine (all Life-781

29

Technologies) and 5% human AB serum (Welsh Blood Transfusion Service). 782

Autofluoresence is adjusted using the analysis software and removed from the final 783

SFC count. 784

785

For analysis of colon leukocyte cytokine production, PBMCs from the colon donor 786

was used as autologous γ-irradiated in an identical manner as that described 787

previously. 1.5 × 105 cells per well and pulsed with peptide pools or DMSO vehicle 788

control for 2 hours @ 37°C. Cells were transferred to a human IFN+/IL-10+ Fluorospot 789

plate with the addition of 5 × 104 cells from peripheral blood and Colon processed 790

tissue with the inclusion of 1/100 human anti-CD28/CD49d co-stimulation antibodies 791

for 18 hours @ 37°C. Plates were assayed and measured as described above. 792

793

Antibody and cytokine measurements 794

Cardiac punctures were performed on CD4-Cre-IL-10flox/flox, CD4-Cre+IL-10flox/flox mice 795

on either naïve mice or after 60 days of MCMV infection and plasma isolated. Plasma 796

was then assayed for anti-Sjögrens Syndrome Antigen IgG by ELISA (Alpha 797

Diagnostics). Samples were analysed according to the manufacturers instructions 798

and Total IgG antibody levels present in the plasma was calculated by interpolation 799

from the calibrator curve provided with the kit. Excised tissue from both Spleen and 800

Salivary Gland (approx. 50-100 mg) were weighed, washed and re-suspended in 801

DMEM. Supernatant was assayed in triplicate for IL-27 p28 production by ELISA and 802

performed according to the manufacturers instructions (eBioscience). 803

804

Viral load quantification 805

Infectious virus quantification was determined using plaque assays as previously 806

described [60]. Viral DNA copy number in saliva was determined using qPCR for 807

relative expression of IE1, as described previously [74]. Oral lavage was performed 808

on the sublingual cavity of anaesthetised mice using 20 µl of sterile PBS. 1 µl of 809

30

sample was used for qPCR and measured using SYBR green (Bio-Rad) using 810

MCMV IE1 forward (5ʹ-AGCCACCAACATTGACCACGCAC-3ʹ) and MCMV IE1 811

reverse (5ʹ-GCCCCAACCAGGACACACAACTC3ʹ) primers. PCR was performed 812

using an MJ Mini Personal Thermal Cycler (Bio-Rad) using the conditions as 813

described previously [74]. To establish a standard curve, DNA copy/µl was 814

determined from the known concentration and molecular weight of the MCMV 815

pARK25 BAC (a kind gift from Alec Redwood, Murdoch University) and assayed for 816

relative IE1 expression as above. 817

818

Macrophage transcriptomics 819

WT, IfnαR-/- and Ifnβ1-/- BM-DM were generated as previously described [60]. Cells 820

were infected with WT-MCMV, MCMVΔIE3 (MOI = 1) or were mock infected [75], and 821

RNA isolated using RNeasy Mini kit at different time-points (Qiagen) according to 822

manufacturer’s instructions. Some cells were stimulated with Poly(I:C) (Invivogen). 823

Quality control (QC) was performed using an Agilent Bioanalyser and, total RNA 824

labeled. RNA was hybridised to Mouse Gene 1.0ST microarrays (Affymetrix) using a 825

WT Expression kit (Ambion, UK). GC metrics of captured data were assessed using 826

Affymetrix Expression Console software and arrays were imported into Partek 827

Genomics Suite (Partek) for downstream analysis. Arrays were normalised using the 828

gcRMA algorithm [76] and data was filtered to include genes with at least 1 signal 829

value of > = 150 across the time course. 830

831

Ethics statement 832

All mice experiments were performed under the UK Home Office project Licence 833

(PPL 30/2969). For human colon and PBMC samples, informed consent was 834

obtained from participants in writing. The Wales Research Ethics Committee granted 835

31

ethical approval for sample use in this study. For use of HCMV sero-positive donor 836

PBMC samples used in this study informed consent was obtained in writing. 837

838

Statistics 839

Power calculations were performed in R using data from a pilot study using CD4-840

Cre/IL-10 mice. It was determined that a minimum of 5 participants per group would 841

be needed to detect a difference in means with 90% power and an alpha value set at 842

0.05. Statistical analysis was performed using the Mann-Whitney U test for paired 843

analysis of viral-load analysis and flow cytometry data. Where more than 2 groups 844

were assessed concurrently (viral-load analysis, ELISA analysis), 1-way ANOVA 845

analysis of data was performed. To assess biological replication: all in vivo 846

experiments were performed multiple times at different times, as stated in figure 847

legends. All outliers were included in datasets, as shown. For all tests performed, p 848

values are reported as *≤0.05, **≤0.01, and ***≤0.001. 849

850

Acknowledgements 851

We acknowledge the NIH tetramer Core Facility for the provision of MHCI 852

biotinylated monomers. We thank Chris Benedict and Chris Hunter for helpful 853

discussions. Our thanks to Konstantinos Kotzamanis for help with the preparation of 854

Figure 7. 855

856

857

858

859

860

861

862

863

32

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865

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1102

1103

1104

1105

1106

1107

1108

1109

41

Supporting Information 1110

1111

S1 Figure. CD4+ IL-10+ T cells are not TH17 or Tr1 cells. 1112

10-BiT reporter mice were infected with MCMV and at day 7 and d14 pi spleen and 1113

salivary glands were isolated. Representative histograms of AhR viable (aqua live/dead-) 1114

Thy1.1- or Thy1.1

+ expression by CD4+CD3+ cells (top), control = fluorescent minus one-1115

stained Thy1.1+ samples. Representative bivariant FACS plot of CD49b/LAG3 viable 1116

(aqua live/dead-) expression by CD4+CD3+ cells (bottom). 1117

1118

S2 Figure. Non-T cells do not produce compensatory IL-10 in CD4-Cre+IL-10flox 1119

mice. 1120


and at day 7 and 14, IL-10 responses in the spleen and salivary glands were measured. 1122

Representative bivariant FACS plots of CD19+ and NK1.1

+ versus IL-10 expression 1123

splenic (top) and salivary gland (d14 bottom) were analysed by viable (aqua live/dead-), 1124

CD19+/CD3

- and NK1.1

+/CD3

- expression. Data is representative of 6 mice per group. 1125

1126

S3 Figure. Increased co-stimulatory ligand expression in the absence of T cell-1127

derived IL-10 during MCMV infection 1128


and at day 7 and 14, myeloid expression in the spleen (A) and salivary glands (B) was 1130

measured. CD11c+MHCII

+ were quantified (left) and % CD80, CD86, OX-40L, 4-1BBL 1131

and CD40 expression (right) was assessed. Data is representative of 5-6 mice per 1132

group. 1133

1134

S4 Figure. MCMV does not induce autoimmunity in the absence of IL-10 1135

production by CD4+ T-cells 1136

42

(A) CD4-Cre-IL-10flox/flox (Cre-) and CD4-Cre+IL-10flox/flox (Cre+) mice were infected with 1137

MCMV and at day 14 and 30 Spleen and Salivary Glands were isolated and 1138

CD4+/DR5+ cells were quantified. Mean ± SEM of total cells from 4-12 mice are 1139

shown and represent 2 separate experiments. (B) Cardiac punctures were performed 1140

d60 pi and anti-SSA IgG was measured by ELISA. Data is representative of 6 naïve 1141

and 16 mice in each group and is representative of 2 experiments. 1142

1143

S5 Figure. MCMV-specific memory CD8+ T-cell inflation is increased in the 1144

absence of IL-27R signaling 1145

Il-27rα-/- or WT mice were infected with MCMV and total virus-specific CD8

+/IFNγ

+ and 1146

CD8+/CD107a

+ T-cells were quantified. Data is presented as mean ± SEM of 5 mice and 1147

is representative of 2 experiments. 1148

1149

S6 Figure. ICOS treatment does not impact on CD4 TH1 responses. 1150

WT (C57BL/6) mice were infected with MCMV and at d6 and d10 pi. 200 g Isotype 1151

control or 200 g ICOS was administered. At d14 pi splenic virus-specific 1152

CD4+/IFN + responses were quantified and expressed at mean + SEM of 6 1153

mice/group. 1154

1155

S7 Figure. Antagonizing of IL-10R and IL-27 during chronicity results in modest 1156

increase in virus-specific IFNγ+CD4+ T cells 1157

WT (C57BL/6) mice were infected with MCMV treated with anti-IL-10R, anti-IL-27, 1158

and/or isotype on day 14 pi, and at d30 pi spleen CD4+/IFNγ+ responses were 1159

quantified and expressed as mean ± SEM of 4 mice/group. 1160

m09 M25 m139 m1420.00

0.05

0.10

0.15

0.20

IL-10IFNγ

IFNγ/IL-10

m09 M25 m139 m1420.00

0.05

0.10

0.15

0.20

IL-10IFNγ

IFNγ/IL-10

m09 M25 m139 m1420.00

0.05

0.10

0.15

0.20

IL-10IFNγ/IL-10

IFNγ

m09 M25 m139 m1420.00

0.05

0.10

0.15

0.20IFNγIL-10IFNγ/IL-10

Spleen

d7 pi d60 pid30 pid14 pi

% C

D4

+

0 10 20 30 600.0

0.5

1.0

1.5m09

M25

m139

m142

0 10 20 30 600.00

0.05

0.10

0.15m09

M25

m139

m142

0 10 20 30 600.00

0.05

0.10

0.15m09

M25

m139

m142

0 10 20 30 600.00

0.05

0.10

0.15

0.20

0.25 m09

M25

m139

m142

% CD4+ IL-10+ % CD4+ IFNγ+

Days post-infection

% C

D4

+

Salivary

Glands

Spleen

B C

D E

F

m09 M25 m139 m1420.00

0.05

0.10

0.15

0.20

0.2512

IL-10IFNγ/IL-10

IFNγ

m09 M25 m139 m1420.0

0.5

1.0

1.5

2.0IFNγIL-10IFNγ/IL-10

m09 M25 m139 m1420.0

0.1

0.2

0.312

IL-10IFNγ/IL-10

IFNγ

m09 M25 m139 m1420.00

0.05

0.10

0.15

0.20

0.2512

IFNγIL-10IFNγ/IL-10

SalivaryGlands

Spleend7

IFNγ

IL-1

0

SalivaryGlands

d14

A medium m09 M25 m139 m142

0.071 0

0

0.24 0

0.032

0.28 0.024

0.072

0.97 0.012

0.049

0.44 0.021

0.021

m1

39

CD4-Cre+

0.069 0.009

0.069

M2

5

0.018 0

0

0.022 0

0.002

0.09 0.003

0.033

0.054 0.012

0.042

0.063 0.02

0.038

0.013 0

0.06

T-Bet

BLI

MP-1

c-M

af0

50

100

150 Thy1.1-

Thy1.1+

*

****

T-Bet

BLI

MP-1

c-M

af0

100

200

300

400

500Thy1.1-

Thy1.1+

***

**ns

Salivary Glands

d14 pi

Spleen

d7 pi

CD

4

Thy1.1

% o

f m

ax s

tain

ing

T-Bet

BLIMP-1

c-Maf

Salivary Glands d14 pi

Spleen d7 piA B

C

Me

dia

n F

luo

rese

nce

In

ten

sity

CD4+ Thy1.1+

CD4+ Thy1.1-

control

12.277.53.189.4

Spleen d7 pi


CX

CR

3

CCR5

CD4+ Thy1.1+

CD4+ Thy1.1-

D

1.11.29

44.65.6

5157.3

4.695.49

14.76.1

17.514.4

1 2 3 40

20

40

60

80

100100

300IFNγ+

IL-10+

IFNγ+IL-10+

1 2 3 40

20

40

60

80

100100

400 IFNγ+

IL-10+

IFNγ+IL-10+

PB

MC

1C

olo

n 1

Healthy Donor CD4+ T cells

0 pp65 gB

IFNγ+

IL-10+

IFNγ+/IL-10+

CRC Total Leukocytes

PB

MC

2C

olo

n 2

PB

MC

3C

olo

n 3

Do

no

r 1

Do

no

r 2

Do

no

r 3

Do

no

r 4

Do

no

r 5

IFNγ+

IL-10+

IFNγ+/IL-10+

0 pp65 gB

pp65

gB

Healthy Donor CD4+ T cellsCRC Total Leukocytes

pp65 gB

CRC Donor ColongBpp65

Donor ID

1 2 3 4 30

50

100

150

200

250IFNγ+

IL-10+

IFNγ+IL-10+

1 2 3 4 50

1

2

3 IFNγ+

IL-10+

IFNγ+IL-10+

SF

C per

10

5 c

ells

A

B

1 2 3 40

20

40

6060

400IFNγ+

IL-10+

IFNγ+IL-10+

1 2 3 40

20

40

60

80

100IFNγ+

IL-10+

IFNγ+IL-10+

Do

no

r 3

targ

ets

on

ly

100

101

102

103

104

105

***

**

CD4-Cre-

CD4-Cre+

CD4 Transfer

IE3 M38 m139 M45

0.0

0.1

0.2

0.3

0.4 ***

***

CD4-Cre-

CD4-Cre+

ns

ns

0.0

0.1

0.2

0.3

0.4**

**

CD4-Cre-

CD4-Cre+

ns

ns

PBS WT IL-10-/- 102

103

104

105

*

*

SG

pfu

/org

an

Lung d60 pi

x1

07 c

ells

ce

lls x

10

5

To

tal C

D8

+ T

etr

am

er+

7 14 28 7 14 21

0 10 20 30 600.000

0.002

0.004

0.006CD4-Cre-

CD4-Cre+

*

0 10 20 30 600.000

0.005

0.010

0.015

0.020CD4-Cre-

CD4-Cre+*

0 10 20 30 600.000

0.002

0.004

0.006

0.008 CD4-Cre-

CD4-Cre+

0 10 20 30 600.000

0.005

0.010

0.015CD4-Cre-

CD4-Cre+*

0 10 20 30 600.000

0.001

0.002

0.003

0.004CD4-Cre-

CD4-Cre+

0 10 20 30 600.000

0.002

0.004

0.006

0.008

0.010CD4-Cre-

CD4-Cre+

****

*

0 10 20 30 600.000

0.001

0.002

0.003

0.004

0.005 CD4-Cre-

CD4-Cre+**

****

0 10 20 30 600.000

0.002

0.004

0.006

0.008CD4-Cre-

CD4-Cre+**

*

***

To

tal S

ple

en

CD

4+ I

FNγ+

ce

lls x

10

7

Days post-infection

m09 M25

m139 m142

m09 M25

m139 m142

Days post -infectionDays post -infection

To

tal S

G C

D4

+ I

FNγ+

ce

lls x

10

4

IE3 M38 m139 M45

A

B

C

D E F

100

101

102

103

104CD4-Cre-

CD4-Cre+ ***

***

Lo

g10 D

NA

co

py/µ

l

28

Spleen d60 pi

SG

pfu

/org

an

Naive D140

50

100

150*

Naive D2 D40

500

1000

1500

2000**

ns

IL-2

7 (

pg

/g tis

su

e)

A BSpleen Salivary Glands

DCs

Mac

roph

age

B-c

ells

Neu

troph

ils

T-cel

ls0

1

2

34

20D0

D2

*

**

*

ns

ns

D14

%IL

-27

+

Spleen

MH

CII

IL-27

CD

11

b

B2

20

Ly6

G

CD

3

Dendritic cells Macrophages B-cells Neutrophils T-cellsC

7.7

12.7

3

0.12

0.13

0.006

0.39

2.33

0

0.63

1.22

0.10

0.47

1.74

0.03

Spleen

d0

Spleen

d2 pi

Spleen

d14 pi

m09 M25 m139 m1420.0000

0.0005

0.0010

0.0015WT

wsx1-/-

CD

4

0.45

0

20

40

60

Thy1.1+Thy1.1-***

ns

gp130

m09 M25 m139 m1420.000

0.001

0.002

0.003

0.004 WT

wsx1-/-

*

**

*

m09 M25 m139 m1420.0000

0.0001

0.0002

0.0003WT

wsx1-/-

m09 M25 m139 m1420.0000

0.0005

0.0010

0.0015

0.0020WT

wsx1-/-

**

**

To

tal C

D4

+ I

L-1

0+

IFNγ

IL-1

0

0.094 0.047

0.31

0.012 0.006

WT

wsx1-/-

Spleen d7 pi

A


Spleen d14 pi x107 Spleen d30 pi x107 Salivary Glands d14 pi x105

Spleen d7 pi

Thy1.1- Thy1.1+


40.7 59.3 78.7 21.3

94.9 5.05 93.7 6.34

%g

p1

30

+

C DB Spleen d7 pi

Spleen d7 pi x107

m09 M25 m139 m1420.0000

0.0005

0.0010

0.0015

0.0020 Isotype+αICOS

m09 M25 m139 m1420.000

0.002

0.004

0.006

0.008Isotype+αICOS

*

*

0.05

ns

Spleen d14 pi x107Salivary Glands d14 pi x105F

To

tal C

D4

+ IL

-10

+

ICOS0

10

20

30

40

Thy1.1-

Thy1.1+

**

%+

ve c

ells


E

CD4+Thy1.1+

CD4+ Thy1.1-

control

% o

f ma

x s

tain

ing

ICOS


G

m09 M25 m139 m1420.000

0.002

0.004

0.006

0.008

0.010 WT

wsx1-/-**

*

***

m09 M25 m139 m1420.000

0.001

0.002

0.003

0.004

0.005WT

wsx1-/-

m09 M25 m139 m1420.0000

0.0005

0.0010

0.0015

0.0020

0.0025WT

wsx1-/-

** *

*

m09 M25 m139 m1420.000

0.002

0.004

0.006

0.008

0.010WT

wsx1-/-

***

100

101

102

103

104 WT

wsx1-/-*

** ***

Lo

g10 D

NA

co

py/µ

l

100

101

102

103

104 WTwsx1-/-

*ns

SG

pfu

/org

an

To

tal C

D4

+ IF

Nγ+

7 14 28Days post-infection

7 14 21 28

Days post-infection

A

B C

Spleen d14 pi x107

Spleen d30 pi x107 Salivary Glands d14 pi x105

Spleen d7 pi x107

DCs

Mac

roph

age

B-c

ells

Neu

troph

ils

T-cel

ls0

5

10

15 + Isotype

+ αIFNαR-1

*

***

***

**

%IL

-27

+WT Virus

WT

IFNB-/-

IFNAR-/-

0 2 4 6 8 10 24Time(hrs)

M

WT

IFNB-/-

IFNAR-/-

WT

IFNB-/-

IFNAR-/-

IE3KO Virus

0 2 4 6 8 10 24Time(hrs)

0 2 4 6 8 10 24Time(hrs)

POLY:IC

LO

G2 F

old

Ch

an

ge

-3.6

3.6

0

A

B

No

rma

lise

d I

L-2

7 e

xp

ressio

n

Poly(I:C)

IFNβ-/-

IFNαR-/-

IFNβ-/-

IFNαR-/-

IFNβ-/-

IFNαR-/-

Isot

ype

+αIL

-10R

+αIL

-27

+αIL

-10R

/αIL

-27

0.000

0.002

0.004

0.006

0.008

0.010 **

**

To

tal C

D4

+ve

IF

Nγ+

ve

ce

lls

Isot

ype

+αIL

-10R

+αIL

-27

+αIL

-10R

/αIL

-27

101

102

103*

**

Lo

g10 D

NA

co

py/µ

l

C3×104 pfu MCMV i.p

d0 d6 p.i

no injection+Isotype i.p

+αIL-27 i.p

+αIL-27 i.p

+αIL-10R i.p

+αIL-10R i.p

no injection

d14 p.i

analysis

E

D

no antibody analysis

analysis

analysis

Spleen d14 pi x107

Me

dia

n F

luo

rese

nce

Inte

nsity

AhR

Spleen d7 p.i Salivary Glands d14 p.i

Salivary Glands d14 p.i

Spleen d7 p.i

CD4 Thy1.1- CD4 Thy1.1+

LAG3

CD

49

b

7.31 0.5

13.4 1.11

7.13 2.22

9.98 0.78

5.36 28.2

2.78 9.16

CD4+ Thy1.1+

CD4+ Thy1.1-

control

IL-10

CD

19

NK

-1.1

CD4-Cre- CD4-Cre+

Spleend7 p.i

Salivary Glandsd14 p.i

48.3 0.2148.3 0.21

CD

19

NK

-1.1

35.4 0.12

0.15 0.07

2.4 0.005 4.02 0.01

0.38 0.2

4.48 0.13 6.09 0.076

2.64 0.78

12.6 0.22 18.1 0.12

1.47 0.54

CD80

CD86

OX40

L

4-1B

BL

CD40

0

5

10

1520

80CD4-Cre-CD4-Cre+

**

**

%+

ve c

ells

CD80

CD86

OX40

L

4-1B

BL

CD40

0

2

4

6

8

1010

30CD4-Cre-CD4-Cre+

**

**

*

**%+

ve c

ells

CD80

CD86

OX40

L

4-1B

BL

CD40

0

5

10

1515

80CD4-Cre-CD4-Cre+

**

%+

ve c

ells

CD80

CD86

OX40

L

4-1B

BL

CD40

0

5

10

15

2020

40CD4-Cre-CD4-Cre+

**

%+

ve c

ells

CD4-Cre- CD4-Cre+0.00

0.02

0.04

0.06

0.08

0.10

CD4-Cre- CD4-Cre+0

2

4

6

8


0.1

0.2

0.3

Spleen

Salivary Gland

CD

11

c+M

HC

II+ ×

10

7


0.05

0.10

0.15

d7 p.i

d14 p.i

d7 p.i

d14 p.i

A

B

CD

11

c+M

HC

II+ ×

10

5

d60 piNaive

0

1000

2000

3000

4000CD4-Cre-

CD4-Cre+

aS

SA

U/m

l


0.0005

0.0010

0.0015

0.0020ns


0.0001

0.0002

0.0003 ns


0.0005

0.0010

0.0015ns


0.0005

0.0010

0.0015 ns

To

tal S

G C

D4

+ D

R5

+ c

ells

x1

05

Spleen Salivary Glands

To

tal S

ple

en

C

D4

+ D

R5

+ c

ells

x1

07

d14 pi

d30 pi

A

B

m139 IE3 M38 M45

0.00

0.05

0.10

0.15

0.20

0.25WT

wsx1-/-

**

**

****

m139 IE3 M38 M45

0.00

0.02

0.04

0.06

0.08

0.10WT

wsx1-/-

**

**

****

To

tal C

D8

+ c

ells x

10

7 IFNγ CD107a

Total Splenic CD4+ve IFNγ+ve

cells x107

Total SG CD4+ve IFNγ+ve

cells x105

m09 M25 m139 m1420.000

0.001

0.002

0.003

0.004Isotype

+αICOS

m09 M25 m139 m1420.0000

0.0001

0.0002

0.0003

0.0004

0.0005Isotype

+αICOS

Isot

ype

+αIL

-10R

+αIL

-27

+αIL

-10R

/αIL

-27

0.000

0.002

0.004

0.006

0.008 Isotype

+αIL-10R

+αIL-27

+αIL-10R/αIL-27

0.2

To

tal C

D4

+ve

IFNγ+

ve

ce

llsSpleen d30 pi x107

Clement et al 141116 PP accepted - COnnecting REpositories · 2017. 2. 8. · 114 [14] and fewer...

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Transcript of Clement et al 141116 PP accepted - COnnecting REpositories · 2017. 2. 8. · 114 [14] and fewer...