cervical cancer-instructed stromal fibroblasts enhance il-23 … · 2019/1/29 · 1 1 cervical...

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Cervical cancer-instructed stromal fibroblasts enhance IL-23 expression in 1

dendritic cells to support expansion of Th17 cells 2

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Barbara Walch-Rückheim1*, Russalina Ströder2, Laura Theobald1, Jennifer Pahne-5

Zeppenfeld3, Subramanya Hegde3, Yoo-Jin Kim4, Rainer Maria Bohle4, Ingolf Juhasz-6

Böss2, Erich-Franz Solomayer2 and Sigrun Smola1 7

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1Institute of Virology and Center of Human and Molecular Biology, Saarland 9

University, Homburg/Saar, Germany 10

2Department of Obstetrics and Gynecology, Saarland University, Homburg/Saar, 11

Germany 12

3Center for Molecular Medicine Cologne and Institute of Virology, University of 13

Cologne, Cologne, Germany 14

4Institute of Pathology, Saarland University, Homburg/Saar, Germany 15

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Running title: 17

Th17 expansion by cancer-instructed DC-fibroblast co-culture 18

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Keywords: 20

Cervical cancer, Human papillomavirus, Interleukin-6, Dendritic cells, IL-23, Th17 21

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Financial support: 23

This work was supported by a grant of the Saarland University to B. Walch-24

Rückheim. B. Walch-Rückheim received founding from the “Else Kröner-Fresenius-25

Stiftung” (2017-A64). 26

Research. on April 13, 2021. © 2019 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 29, 2019; DOI: 10.1158/0008-5472.CAN-18-1913

http://cancerres.aacrjournals.org/

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*Corresponding author: 27

Dr. Barbara Walch-Rückheim 28

Institute of Virology, Saarland University 29

Kirrbergerstrasse, Building 47 30

D-66421 Homburg/Saar, Germany 31

Phone: ++49-6841-1623405 32

Fax: ++49-6841-1623980 33

E-mail: [email protected] 34

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The authors declare no potential conflicts of interest. 37

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Word count: 5674 words 39

Total number of figures and tables: 40

7 Figures, 6 Supplementary Figures, 1 Supplementary Table 41

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mailto:[email protected]


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Abstract: 43

Persistent infection with high-risk human papillomavirus (HPV) is a prerequisite for 44

the development of cervical cancer. HPV-transformed cells actively instruct their 45

microenvironment, promoting chronic inflammation and cancer progression. We 46

previously demonstrated that cervical cancer cells contribute to T-helper-17 (Th17) 47

cell recruitment, a cell type with pro-tumorigenic properties. In this study we analysed 48

the expression of the Th17-promoting cytokine Interleukin(IL)-23 in the cervical 49

cancer micromilieu and found CD83+ mature dendritic cells (mDC) co-expressing IL-50

23 in the stroma of cervical squamous cell carcinomas in situ. This expression of IL-51

23 correlated with stromal Th17 cells, advanced tumor stage, lymph node 52

metastasis, and cervical cancers recurrence. Co-cultures of cervical cancer-53

instructed mDC and cervical fibroblasts led to potent pro-tumorigenic expansion of 54

Th17 cells in vitro but failed to induce anti-tumor Th1 differentiation. Correspondingly, 55

cervical cancer-instructed fibroblasts increased IL-23 production in co-cultured 56

cervical cancer-instructed mDC, which mediated subsequent Th17 cell expansion. In 57

contrast, production of the Th1-polarizing cytokine IL-12 in cancer-instructed mDC 58

was strongly reduced. This differential IL-23 and IL-12 regulation was the 59

consequence of an increased expression of IL-23 subunits IL-23p19 and IL-12p40 60

but decreased expression of the IL-12 subunit IL-12p35 in cervical cancer-instructed 61

mDC. Cervical cancer cell-derived IL-6 directly suppressed IL-12p35 in mDC but 62

indirectly induced IL-23 expression in fibroblasts-primed mDC via CAAT/enhancer-63

binding protein (C/EBP)-dependent induction of IL-1. In summary, our study 64

defines a mechanism by which the cervical cancer micromilieu supports IL-23-65

mediated Th17 expansion associated with cancer progression. 66

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Introduction: 68

Cervical carcinogenesis is a consequence of persistent infection with genital high-risk 69

human papillomavirus (HPV). Cervical cancer develops through cervical 70

intraepithelial neoplasia (CIN1-3) in a well-characterized multi-step process that 71

takes years or decades. 72

As a mechanism of immune escape the high-risk HPV oncoproteins interfere with 73

inflammatory signaling pathways in keratinocytes. They suppress the expression of 74

chemokines, including monocyte chemoattractant protein-1 (CCL2) and macrophage 75

inflammatory protein-3α (CCL20), eventually preventing the recruitment of antigen-76

presenting cells to the epithelium (1-3). Thus, inflammatory responses are rarely 77

observed in persisting low-grade lesions. 78

At later stages of cervical carcinogenesis, pre-cancerous high-grade lesions and 79

invasive cervical cancers are often associated with strong inflammatory infiltrates in 80

the stroma (4-10). Different studies have shown that HPV-transformed keratinocytes 81

actively contribute to the inflammatory microenvironment during cervical 82

carcinogenesis via production of the cytokine IL-6 (6,8,9,11). IL-6 is expressed in 83

high-grade as well as invasive lesions (12). It preferentially acts in a paracrine 84

manner at later stages of the disease (12) to suppress immune functions in myeloid 85

immune cells, such as CCR7-dependent migration of DC but enhances their tumor-86

promoting activities, such as MMP-9 production (6-8). Furthermore, we recently 87

demonstrated that cervical cancer cells actively contribute to the recruitment of Th17 88

cells. While HPV-infected keratinocytes express only low amounts of the Th17-89

attracting chemokine CCL20 (2,9), they instruct cervical fibroblasts to produce 90

CCL20 in an IL-6/CAAT/enhancer-binding protein (C/EBP)-dependent manner 91

and thereby support Th17 cell recruitment (9). Cervical cancer-infiltrating Th17 cells 92




5

in situ were correlated with advanced International Federation of Gynecology and 93

Obstetrics (FIGO) (13) stages of the tumors (9) and a poor prognosis for the patients 94

(14). 95

Th17 cells were identified as a sub-group of T-helper cells (15) exhibiting pro-96

inflammatory as well as tumor-promoting properties in different cancer types (16,17). 97

Human Th17 cell differentiation from naïve T cells is dependent on the cytokines IL-98

6, TGF-, IL-1 and IL-23 (18,19). IL-23 has been described to be necessary for the 99

expansion, activity and cytokine release of Th17 cells (20). IL-23 is a member of the 100

IL-12 family which has the ability to modulate T-cell activity (21). It is a heterodimeric 101

cytokine mainly expressed by DC containing the subunit IL-23p19 and shares the 102

subunit IL-12p40 with IL-12, a cytokine crucial for Th1 polarization (20). Similar to IL-103

17, serum levels of IL-23 were shown to be increased in cervical cancer patients 104

(22). 105

Previously, we detected CD83-positive DC in cervical cancer biopsies and 106

demonstrated that cervical cancer cells suppress their CCR7 expression (8), thus 107

promoting the accumulation of phenotypically mature but functional impaired DC in 108

the tumor stroma (8). In the peripheral tissue, DC are in permanent contact with their 109

tissue microenvironment interacting with stromal cells, i.e. fibroblasts. Fibroblasts can 110

influence DC functions and their cytokine and chemokine secretion patterns (23-25). 111

In this study we investigated the mechanism underlying the expansion of Th17 cells 112

in cervical cancers. We demonstrate that the presence of CD83+IL-23p19+ DC in 113

cervical cancers in situ correlate with the presence of tumor-infiltrating Th17 cells. 114

Our data provide evidence that cervical cancer cells differentially regulate the 115

expression of the T cell polarizing cytokines IL-23 and IL-12 in DC. While cervical 116




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carcinoma cell-derived IL-6 suppressed the IL-12 expression by inhibition of the IL-117

12p35 subunit in DC, it induced the expression of C/EBP-mediated IL-1 production 118

of fibroblasts. As a consequence fibroblasts produced IL-1 induced the expression 119

of the Th17-expanding cytokine IL-23 by cervical cancer-instructed DC. This led to 120

the expansion of memory CD4+IL-17+ T cells in an IL-23-dependent manner. To our 121

knowledge, this is the first report on a mechanism how cervical cancer cells can 122

support the expansion of Th17 cells. 123

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Material and methods: 125

Ethics statement 126

This study has been conducted according to Declaration of Helsinki principles. IHC 127

and IF stainings of anonymized tissue samples and the usage of primary human 128

cervical fibroblasts or PBMC were approved by the Ethics Committees of the Medical 129

Faculty of the Saarland University at the Saarland Ärztekammer. Written informed 130

consent was provided by all study participants. 131

132

Tissue specimens, immunohistochemical and Immunofluorescence analysis 133

Formalin fixed paraffin-embedded anonymized lesions of the cervix uteri from 35 134

patients were taken from the local pathology archive of the Saarland University 135

Medical Center. Histological classification was ascertained by expert pathologists 136

(YJK or RMB). Lesions were stained with pan-cytokeratin Ab and co-stained for CD4 137

and IL-17, CD83 and IL-23p19 or -smooth muscle actin (-SMA) and IL-1 or CD83 138

as described in Supplementary Materials and Methods. To evaluate the number of 139

infiltrating Th17 cells or CD83+IL-23p19+cells five randomized pictures (200x) were 140

taken per biopsy and the number of CD4+IL-17+ or CD83+IL-23p19+ cells was 141

counted. 142

143

Cell culture and collection of conditioned media 144

HPV18-positive cervical carcinoma cell lines SW756 (ATCC CRL-10302), HeLa 145

(ATCC CCL-2) and HPV16-positive SiHa (ATCC HTB-35) and CaSki (ATCC CRL-146

1550) obtained from M. von Knebel-Doeberitz before 2000 were authenticated by 147

qRT-PCR for HPV16 or HPV18 E6 and E7 expression and by the German collection 148

of microorganisms and cell cultures (DSMZ) using STR DNA typing in May 2017. 149

Normal exocervical keratinocytes (NECK), normal exocervical fibroblasts (NECF) and 150




8

fibroblasts from cervical cancer outgrowth cultures were cultured as previously 151

described (9,26,27). Cell lines were passaged for less than 3 month. NECF were 152

used in passage 2 after isolation. Fibroblasts from cervical cancer outgrowth cultures 153

were directly used without passaging. The isolated fibroblasts were characterized by 154

qPCR and immunofluorescence by evaluation of stromal, epithelial, endothelial and 155

immune cell markers (Supplementary Materials and Methods; Supplementary 156

Figure S1ABCD and S2AB). For conditioned media cells were cultured at a density of 157

1x106/ml. After 24 h, fresh RPMI 1640 medium (Sigma) plus supplements (10% heat-158

inactivated endotoxin-tested fetal calf serum (Biochrom, Berlin, German) and 1 mM 159

sodium pyruvate) was added. Conditioned media were collected 24 h later. 160

161

Stimulation of fibroblasts with conditioned media of cervical cancer cells, IL-6 162

neutralization and transfections 163

NECF were stimulated for 24 hours with conditioned media of cervical cancer cells or 164

NECK or plain medium as a control. Cells were washed and cultured again in fresh 165

RPMI. 24 h later, supernatants were collected. Neutralizing anti-IL-6 antibody or 166

matched isotype control antibody (10 µg/ml, R&D Systems, South Beloit, IL) was 167

added to conditioned media 30 minutes or 2 h before usage as indicated. Ten pmol 168

of indicated siRNAs (ON-TARGETplus Non-targeting siRNA #2 and ON-TARGET 169

smart-pool for C/EBP, all from Thermo Fisher Scientific) were transfected with 170

Lipofectamine RNAiMax (Invitrogen). 24 h posttransfection, cells were stimulated as 171

described above. 172

173

Generation of dendritic cells and co-culture experiments 174

Monocytes were isolated out of whole blood from healthy donors as previously 175

described (8). To generate cervical-cancer-instructed DC (8) or fibroblast-instructed 176




9

DC, monocytes were cultured at a density of 1x106 cells/ml for 6 days in six‐well 177

plates in RPMI medium plus supplements, GM‐CSF (100 ng/ml Leukine; Berlex, 178

Montville, NJ) and IL‐4 (5 ng/ml; Miltenyi Biotech). Conditioned media from cervical 179

cancer cells or from fibroblasts from cervical cancer outgrowth cultures (final dilution 180

of 50% vol/vol) or recombinant human IL‐6 (100 ng/ml, PeproTech) were added from 181

the first day of culture. Cells were fed every two days with medium, conditioned 182

media or rhIL-6. In neutralization experiments, neutralizing anti-human IL-6 183

monoclonal or isotype-control antibody (10 µg/ml, R&D Systems) was added to 184

conditioned media 30 min before usage. DC maturation was induced with tumor 185

necrosis factor- (TNF- , 500 U/ml; Bender & Co,Vienna, Austria) on day 6 for 8 h, 186

followed by three washing steps and used for co-culture experiments. 187

For co-culture experiments, NECF were seeded at a density of 1x106 cells/ml. In 188

same experiments NECF were pre-stimulated with conditioned media of cervical 189

cancer cells. After 24 h 1x106 cells/ml normal or cervical cancer-instructed immature 190

or mature DC were added. As a control, DC and fibroblasts were cultured alone 191

under the same conditions. After 16 h supernatants were harvested and cells were 192

collected for flow cytometry analysis or RNA isolation. For cell separation DC were 193

removed from adherent fibroblast monolayers. DC and fibroblasts were washed three 194

times with PBS. Purity of DC was determined by flow cytometry using the DC specific 195

marker CD1a and was 86.7% 7.2 in averaged three independent experiments. To 196

control purity of fibroblasts and to exclude contamination with DC, fibroblasts were 197

stained with CD1a. Fibroblasts showed no expression of the DC specific marker. In 198

neutralizing experiments, neutralizing anti-human IL-1 monoclonal or isotype-control 199

antibody (2 µg/ml, R&D Systems) was added to supernatants of pre-stimulated 200

NECF 2 h before co-culture. 201




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202

Quantitative real-time PCR 203

RNA isolation, cDNA synthesis, real-time PCR and normalization to -Actin or 204

RPL13A were performed as previously described (9,26). The 75-bp fragment of IL-205

12B was detected with primers 206

5′-CCCTGACATTCTGCGTTCA-3′ and 5′-AGGTCTTGTCCGTGAAGACTCTA-3′ and 207

probe no. 37; the 91-bp fragment of IL-12A with primers 208

5′-CACTCCCAAAACCTGCTGAG-3′ and 5′-CAATCTCTTCAGAAGTGCAAGG-3′ and 209

probe no. 50; the 71-bp fragment of IL-23A with primers 210

5′-AGCTTCATGCCTCCCTACTG-3′ and 5′-CTGCTGAGTCTCCCAGTGGT-3′, and 211

probe no. 30 and the 70-bp fragment of IL-1 with primers 212

5`-CTGTCCTGCGTGTTGAAAGA and 5`-TTGGGTAATTTTTGGGATCTACA and 213

probe no. 78. 214

215

ELISA 216

IL-12p70, IL-23 and IL-1 concentrations were determined with DuoSet (R&D 217

Systems) according to the supplier`s instructions. Detection limits were 31.25 pg/ml, 218

125 pg/ml or 3.91 pg/ml, respectively. 219

220

T cell isolation, T cell stimulation experiments and IL-23 neutralization 221

Naïve or memory CD4+ T cells were isolated by negative selection from fresh PBMC 222

using human Naïve CD4+ T Cell Isolation Kit II or Memory CD4 T Cell Isolation Kit 223

(Miltenyi Biotech), respectively. Purity was determined with anti-CD4, anti-CD45RO 224

and anti-CD45RA and was 90.2% 4.2 for naïve and 92.8% 5.7 for memory T cells 225

in averaged three independent experiments. Cells were cultured in RPMI 1640 226




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medium supplemented with 10% heat-inactivated endotoxin-tested FCS (Biochrom, 227

Berlin, Germany). 228

Supernatants from DC-NECF co-cultures (final dilution of 50% vol/vol) or 100 ng/ml 229

rhIL-23 (Miltenyi Biotech) as a control were added to 1x105 naïve or memory CD4-230

positive T cells/96-well from the first day of culture in the presence of beads from the 231

T-cell Activation/Expansion Kit (Miltenyi Biotech). Cells were fed every two days with 232

co-cultures supernatants or rhIL-23. In neutralization experiments, anti-human IL-23 233

or isotype-control antibody (1 µg/ml, R&D Systems) was added to co-culture 234

supernatants 30 min before usage. On day 6, T cells were re-stimulated with 235

Phorbol-12-myristate-13-acetat (PMA; 5 ng/ml)/ionomycin (500 ng/ml) (both from 236

Sigma) for 6 h. After 2 h brefeldin A (10 µg/ml; Sigma) was added. Supernatants 237

were collected and analyzed by ELISA and cells were stained for flow cytometry 238

analysis. 239

240

Cell staining, flow cytometry analysis and determination of Th17 numbers 241

Stimulated T cells were treated with 2 mM EDTA for 15 min to disrupt cell-cell 242

interactions, fixed with 3% paraformaldehyde and stained under permeabilizing 243

conditions using anti-CD4-FITC, anti-IL-17-APC and anti-IFN--BV421 or anti-CD4-244

APC and IFN--FITC or respective conjugated isotype-control antibodies (BD 245

Biosciences and Miltenyi Biotech) and analyzed by flow cytometry (FACSCantoII; BD 246

Biosciences). Numbers of Th17 cells were determined using Trucount™ Absolute 247

Counting Tubes (BD Biosciences) according to the manufacturer`s suggestions. 248

249

Statistical analysis 250




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All statistical analyses were performed using the GraphPad Prism 5 (GraphPad 251

Software, CA, USA) program. To evaluate the statistical differences between the 252

analyzed groups, a two-sided t-test was applied for the comparison between 2 253

groups, the one-way analysis of variance test (with Bonferroni posttest) for 254

comparison of >2 groups. Significances are indicated by asterisks 255

(*<0.05;**<0.01;***<0.001). Correlation between the number of CD83+IL-23p19+ cells, 256

Th17 cells and FIGO (13) stages of squamous cell carcinomas (SCC) was done 257

using Spearman rank correlation. 258

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Results: 260

CD83/IL-23p19 positive DC correlate with Th17 cells in cervical cancers in situ. 261

35 cervical cancer biopsies were stained for CD83/IL-23p19 and CD4/IL-17 (Th17 262

cells), respectively using double immunofluorescence (Figure 1A; isotype control 263

stainings in Supplementary Figure S3). 76% of IL-23p19+ cells were also positive for 264

CD83. The absolute numbers of CD83+IL-23p19+ cells significantly correlated with 265

the presence of Th17 cells in the stroma (Figure 1B; r=0.5100; p=0.0040) while the 266

correlation between total numbers of CD83+ cells and Th17 cells was lower 267

(Figure 1C; r=0.4413; p=0.0080) and no correlation was between the numbers of 268

CD83-/IL-23p19+ cells and Th17 cells (Figure 1D; r=-0.09848; p=0.6787). The 269

strongest correlation was observed when we evaluated the percentage of IL-23p19-270

expressing CD83+ cells per total CD83+ cells and percentage of IL-17-expressing 271

CD4+ cells per total CD4+ cells (Figure 1E; r=0.6946; p0.0001). Moreover, the 272

presence of tumor-infiltrating IL-23p19-expressing CD83+ and Th17 cells both 273

significantly correlated with more advanced FIGO stages of the respective biopsies 274

(Figure 1FG) and patients with lymph node metastasis showed significantly more 275

CD83+IL-23p19+ or Th17 cells (Figure 1HI). Notably, retrospective analysis 276

demonstrated that in patients developing recurrence of cervical cancers IL-23p19-277

expressing CD83+ cells per total CD83+ cells (Figure 1J) and IL-17-expressing CD4+ 278

cells per total CD4+ cells (Figure 1K) were significantly more frequent in their tumor 279

tissues than in patients without relapse. Thus the results from our in situ analysis 280

indicated that the IL-23 expression of mature CD83+ DC might be linked to enhanced 281

proportions of IL-17-producing CD4+ T cells within total CD4+ cells during cervical 282

cancer progression. 283

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Cervical fibroblasts increase the IL-23 but not IL-12 expression in cervical 285

cancer-instructed DC. 286

To analyze the IL-23 regulation in DC in the cervical tumor micromilieu in vitro, 287

monocytes were cultivated for six days with IL-4 and GM-CSF in the absence or 288

presence of conditioned media of different HPV16-positive (SiHa and CaSki) or 289

HPV18-positive (SW756 and HeLa) cervical cancer cells to generate normal or 290

cancer-instructed DC (8). To induce DC maturation cells were stimulated with TNF- 291

for 8 h (Figure 2A). Since 77.6% of CD83+ DC were in contact with stromal cells in 292

the cervical tumor micromilieu interacting with -SMA positive myofibroblasts in our 293

in situ analysis (Figure 2B), we analyzed the impact of fibroblast on the IL-23 294

expression in co-cultures of mDC and NECF. 295

Normal and cervical cancer-instructed immature DC produced low levels of IL-23 (up 296

to 171 pg/ml). Stimulation with TNF- alone induced the expression of IL-23 in 297

normal and in cervical cancer-instructed DC (up to 556 pg/ml). The addition of NECF 298

to DC for 16 h significantly increased the IL-23 secretion in co-cultures with normal 299

and cervical cancer cell-instructed mDC (up to 3092 pg/ml; Figure 2C; grey bars). 300

Interestingly, when we used fibroblasts that were pre-stimulated with conditioned 301

media of cervical cancer cells, the IL-23 expression was further increased in mDC-302

fibroblasts co-cultures (up to 6878 pg/ml; black bars). Notably, a significantly 303

enhanced IL-23 expression we found in co-cultures of normal mDC and fibroblasts 304

from cervical cancer outgrowth cultures (black bars) in comparison to co-cultures with 305

NECF (grey bars) (Figure 2D). 306

In contrast to IL-23, the Th1-polarizing cytokine IL-12 was regulated in an opposite 307

manner. Normal and cervical cancer-instructed immature DC produced very low 308




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levels of IL-12 (up to 32 pg/ml). TNF- stimulation induced the IL-12 expression in 309

normal DC up to 500 pg/ml (white bar) and the addition of NECF (grey bar) or pre-310

stimulated fibroblasts (black bar) significantly induced the IL-12 expression in co-311

cultures with normal mDC (Figure 2E). In contrast, the IL-12 expression was 312

significantly lower in cervical cancer-instructed mDC (102-140 pg/ml; white bars) and 313

neither the addition of NECF (grey bars) nor of pre-stimulated fibroblasts (black bars) 314

increased the IL-12 expression in co-cultures with cancer-instructed mDC (216 to 315

396 pg/ml). A similarly differential IL-23 versus IL-12 regulation pattern was obtained 316

after using the TLR4 agonist LPS as a DC maturation stimulus 317

(Supplementary Figure S4AB). 318

To investigate whether efficient DC maturation took place in the presence of NECF, 319

the expression of maturation markers was analyzed by flow cytometry. After TNF- 320

or LPS stimulation CD83, CD80, CD86, MHC I and MHC II were up-regulated in 321

normal and to similar levels in cancer-instructed mDC. Co-cultivation with fibroblasts 322

had no impact on maturation marker expression but slightly enhanced the MHC I 323

expression (Supplementary Table S1). In summary, we found that co-culture of 324

normal mDC with NECF enhances IL-12 and IL-23 expression. Cervical cancer cells 325

however, selectively interfere with IL-12 expression and rather promote IL-23 326

expression in mDC-fibroblasts co-cultures. 327

328

Cervical cancer cells interfere with IL-12 but not IL-23 expression in mature DC 329

by suppression of the IL-12p35 subunit. 330

To study the differential regulation of IL-12 and IL-23, DC and NECF were separated 331

after co-cultivation and mRNA was prepared from both cell types. As expected, DC 332

were the main producers of IL-12p35, IL-12p40 and IL-23p19 transcripts in DC-NECF 333




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co-cultures but with varying expression levels (Figure 3ABC). Expression of the 334

IL-23p19 subunit was detectable in normal and in cervical cancer-instructed mDC 335

after co-culture with NECF (up to 2.4-fold expression; grey bars). The expression 336

was further increased in mDC after co-culture with fibroblasts pre-stimulated with 337

conditioned media of cervical cancer cells (Figure 3A, up to 5.8-fold; black bars). The 338

same expression pattern in mDC was found for the IL-12p40 subunit shared by IL-12 339

and IL-23 (Figure 3B). However, after co-culture, IL-12p35 production was only 340

induced in normal mDC. In contrast, the expression of the IL-12p35 subunit was 341

significantly reduced in cervical cancer-instructed mDC, independently of whether 342

they were co-cultured with NECF or with pre-stimulated fibroblasts (Figure 3C; up to 343

97.4 % reduction). Similarly results concerning IL-23p19, IL-12p40 and IL-12p35 344

expression were obtained after using the TLR4 agonist LPS as a DC maturation 345

stimulus (Supplementary Figure S4CDE). In conclusion, co-cultivation of fibroblasts 346

with mDC increase the IL-23p19 and IL-12p40 expression in DC, but cervical cancer 347

cells interfere with IL-12 production by suppression of the IL-12p35 subunit. 348

349

IL-6 produced by cervical cancer cells reduces the IL-12 expression via IL-350

12p35 suppression in mDC. 351

Next we were interested in the nature of the soluble factors present in the 352

conditioned media of cervical cancer cells suppressing the IL-12 expression in mDC. 353

We recently demonstrated that cervical cancer cell-derived IL-6 suppresses the 354

transcription factor NF-B in phenotypically mature DC (8). Since NF-B is a 355

regulator of IL-12p35 (28) and cervical cancer cells express high amounts of IL-6 in 356

vitro (9,12), the cytokine IL-6 was regarded as an interesting candidate. In mDC, 357

differentiated in the presence of increasing amounts of recombinant human IL-6 358




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(rhIL-6) (6.25-100 ng/ml), the IL-12 expression was dose-dependently suppressed on 359

protein level after co-culture with NECF (Figure 4A; up to 96 %). Furthermore, after 360

separation of the cells the IL-12p35 subunit was dose-dependently suppressed by 361

rhIL-6 on mRNA level in mDC (Figure 4B; up to 77.8 %). While IL-12p19 was not 362

affected by rhIL-6, IL-12p40 was reduced to a lesser extent (35 % reduction) than 363

p35 (Figure 4CD). IL-6 neutralization in the conditioned media of SW756 and SiHa 364

cells, which express up to 3586 pg/ml of IL-6 (Figure 4E), completely restored the IL-365

12 expression of cervical cancer-instructed mDC co-cultured with NECF (Figure 4F) 366

while the IL-23 expression was not affected (Figure 4G). Taken together, our results 367

clearly showed that IL-6 is the crucial factor for the cervical cancer cell-mediated IL-368

12 suppression in cancer-instructed mDC. 369

370

Cervical cancer cells induce IL-1 expression in fibroblasts in an IL-6/C/EBP-371

dependent manner which mediates the enhanced IL-23 expression in mDC. 372

Next we were interested in the mechanism of increased IL-23 expression in mDC 373

mediated by cervical cancer-instructed fibroblasts. Stimulation of NECF with 374

conditioned media from different cervical cancer cells (a scheme of the stimulation 375

experiments is presented in Figure 5A) resulted in up to 8.7-fold enhanced 376

expression of the fibroblast activation marker -SMA (Supplementary Figure S1D) 377

and lead to potent induction (up to 9.4-fold; Figure 5B) of the cytokine IL-1, a 378

potential inducer of IL-23 expression (29), reaching levels up to 555 pg/ml 379

(Figure 5C). No significant induction was observed after stimulation with 380

supernatants derived from normal HPV-negative NECK. 381




18

We have recently shown that cervical cancer cell-derived IL-6 induces the C/EBP 382

pathway in cervical fibroblasts (9), a regulator of IL-1 expression (30,31). NECF 383

expressed the IL-6 receptor (IL-6R) and rhIL-6 induced IL-1 production and C/EBP 384

expression in NECF (Supplementary Figure S5ABC). To elucidate the impact of IL-385

6/C/EBP-dependent signaling on IL-1 induction, IL-6 neutralizing antibodies and 386

C/EBP-specific siRNAs were used. IL-6 neutralization in the conditioned media of 387

cervical cancer cells reduced C/EBP expression (Supplementary Figure S5D) and 388

led to a significant reduction of IL-1 protein production by cervical cancer-instructed 389

NECF (77.8 %-80.5 % reduction, Figure 5D). Furthermore, knockdown of C/EBP, 390

which was confirmed by Western blot analysis (Figure 5E, 77.9 % reduction), 391

significantly reduced IL-1 production induced by cervical cancer cells in NECF 392

(Figure 5F, up to 81.1 % reduction). To analyze the relevance of IL-1 produced by 393

cervical cancer-instructed fibroblasts for the IL-23 induction of mDC, pre-stimulated 394

fibroblasts were incubated with neutralizing IL-1 antibodies. Neutralization of IL-1 395

significantly reduced the IL-23 expression in co-cultures with normal and cervical 396

cancer-instructed mDC (Figure 5G, 76 % reduction). 397

CD83+ mDC were in contact with stromal -SMA-positive myofibroblasts in cervical 398

cancer tissues (Figure 2B). To evaluated the impact of freshly isolated fibroblasts 399

from cervical cancer outgrowth cultures ex vivo on the IL-23 expression of mDC, we 400

generated DC in the presence of fibroblasts supernatants for six days (fibroblast-401

instructed DC) or cervical cancer-instructed DC. Freshly isolated cancer-instructed 402

fibroblasts from three different donors released significantly higher amounts of IL-6 403

(Figure 5H; 3951 pg/ml) and IL-1 (Figure 5I; 537 pg/ml) protein in their supernatants 404

in comparison with NECF (55 pg/ml or 19.5 pg/ml, respectively). Neither IL-6 405




19

neutralization in the conditioned media used for generation of fibroblasts-instructed or 406

cervical cancer-instructed DC nor during co-cultures with fibroblasts from cervical 407

cancer outgrowth cultures did affect IL-23 production by mDC (Figure 5J). However, 408

neutralization of IL-1 during co-culture of fibroblasts-instructed DC and fibroblasts 409

from cervical cancer outgrowth cultures significantly reduced IL-23 production by 410

mDC (Figure 5K). In contrast, supernatants of fibroblasts from cervical cancer 411

outgrowth cultures reduced the IL-12 expression in mDC and neutralization of IL-6 in 412

fibroblast supernatants significantly restored the IL-12 expression (Supplementary 413

Figure S5E). 414

Taken together, our data clearly showed that cervical cancer cell- and cancer-415

associated fibroblast-derived IL-6 is a crucial factor mediated the reduced IL-12 416

expression in cervical cancer- and fibroblasts-instructed DC. Furthermore, cervical 417

cancer cell-derived IL-6 induced C/EBP-dependent IL-1 expression in NECF. IL-1 418

produced by cancer-instructed NECF or by primary fibroblasts from cervical cancer 419

outgrowth cultures mediated the enhanced IL-23 production in co-cultured normal 420

and cervical cancer-instructed mDC. 421

To validate our in vitro results in vivo, we stained SCC biopsies with a pan-422

cytokeratin Ab and for IL-1 and -SMA. Indeed, our in situ analysis revealed -423

SMA-positive myofibroblasts (green) in the stroma that coexpressed IL-1 (red) 424

(Figure 5L, upper panel) and CD83-positive cells (green) coexpressing IL-23p19 425

(red) in the same area of the tumor biopsy (lower panel) confirming our in vitro 426

results. 427

428




20

Cervical cancer-instructed mDC-fibroblasts co-cultures promote Th17 cell 429

expansion via IL-23. 430

Next we studied the impact of IL-23 produced by cervical cancer-instructed mDC-431

fibroblasts co-cultures on the expansion of Th17 cells. CD4+ memory T cells were 432

cultured with CD2/CD3/CD28 beads in the presence of supernatants from mDC-433

fibroblasts co-cultures for 6 days (Figure 6A). Intracellular flow cytometry analysis 434

revealed increased frequencies of IL-17-producing CD4+ memory T cells under 435

influence of rhIL-23 or supernatants of normal (Figure 6B, upper panel) as well as 436

cervical cancer-instructed mDC-NECF co-cultures (Figure 6B, lower panel). 437

CD4+/IFN-+ (Th1) cells were only slightly, IL-17+/IFN-+ double positive CD4+ T cells 438

not affected by co-culture supernatants (Figure 6C). Using Trucount Absolute 439

Counting Tubes we found enhanced Th17 numbers (up to 2.5-fold increase by co-440

mDC-NECF culture supernatants in comparison to medium control) which were 441

further enhanced (up to 2.1-fold) by supernatants from normal or cervical cancer-442

instructed mDC co-cultured with pre-stimulated fibroblasts (Figure 6D). Furthermore, 443

we found significantly elevated IL-17A protein levels (up to 3-fold) in the supernatants 444

of CD4+ memory T cells cultured with rhIL-23 or mDC-fibroblasts supernatants in 445

comparison to medium control (Figure 6E; normalized to 1000 Th17 cells). Highest 446

IL-17A amounts were produced by CD4+ memory T cells under the influence of co-447

culture supernatants from mDC and pre-stimulated fibroblasts (up to 2108 pg/ml). 448

Thus, our results show that cervical cancer-instructed mDC-fibroblasts co-cultures 449

enhanced both, numbers of Th17 cells as well as the IL-17A production per T cell. 450

Supernatants from mDC-fibroblasts co-cultures or rhIL-23 slightly induce IL-17 451

expression in CD4+ naïve T cells (1.6-fold; Supplementary Figure S6A). 452

Supernatants of NECF and normal mDC co-cultures or rhIL-12 as a control induced 453




21

IFN- production in naïve CD4+ T cells after 6 d of culture (Supplementary 454

Figure S6B; 28 % double-positive cells), but in naïve CD4+ T cells cultured in 455

supernatants from NECF and cervical cancer-instructed mDC co-cultures the IFN- 456

production was significantly reduced (64.5 % reduction). 457

To analyze the relevance of IL-23 from mDC-fibroblasts co-cultures for Th17 458

expansion, we neutralized IL-23 in supernatants of co-cultures with normal as well as 459

cervical cancer-instructed mDC and fibroblasts. Anti-IL-23 antibodies significantly 460

reduced the frequencies (76% decrease; Figure 6F) and numbers of Th17 cells 461

(85.2% decrease; Figure 6G) as wells as IL-17A protein production by Th17 cells 462

(96.7 % reduction, Figure 6H), which was not observed with the respective isotype 463

control antibodies. 464

To investigate the relevance of IL-1 produced by stimulated fibroblasts or ex vivo by 465

fibroblasts from cervical cancer outgrowth cultures for the enhanced IL-23 production 466

in DC and subsequent IL-23-dependent Th17 expansion we neutralized IL-1 in 467

fibroblast supernatants. Neutralization of fibroblast-derived IL-1, and not CCL20 468

(Supplementary Figure S6CDEF) resulted in significant reduced Th17 frequencies 469

(Figure 6I) and numbers (Figure 6J) and per T cell IL-17A protein production 470

(Figure 6K). In conclusion, our data showed that cervical cancer-instructed mDC-471

fibroblasts co-cultures support IL-17 production of CD4+ memory T cell in an IL-23-472

dependent manner. 473

474




22

Discussion: 475

Th17 cells, a T cell subset with pro-tumorigenic properties, infiltrate cervical cancers 476

and correlate with advanced tumor stages (9,32). We previously described that 477

cervical cancer cells actively contribute to Th17 cell recruitment during cervical 478

cancer progression, instructing cervical fibroblasts to produce the Th17-479

chemoattractant protein CCL20 (9). 480

In this study we investigated a novel mechanism underlying Th17 cell expansion. Our 481

in situ analysis identified CD83+ mature DC in cervical cancer tissues expressing the 482

IL-23 subunit p19. Notably, CD83+IL-23p19+ cells significantly correlated with stromal 483

Th17 cells and the severity of the disease. We show that co-cultivation of cervical 484

cancer-instructed mDC with cervical fibroblasts enhanced the IL-23 expression of 485

mDC and pre-stimulation of fibroblasts with conditioned media of cervical cancer 486

cells further increased the IL-23 expression. In contrast, the IL-12 expression of 487

cervical cancer-instructed mDC was strongly reduced. We identified cervical cancer 488

cell-derived IL-6 as the central mediator of the differential expression of IL-23 and IL-489

12. We show that cervical cancer cells induced C/EBP-dependent IL-1 expression 490

via IL-6 in fibroblasts. IL-1 we identified as inducer of the IL-23 expression of mDC 491

in mDC-fibroblasts co-cultures. Freshly isolated cancer-instructed fibroblasts 492

supported the increased IL-23 and reduced IL-12 expression of mDC by secretion of 493

IL-6 and IL-1 ex vivo. In line with previous results, IL-6 suppressed IL-12 production 494

by targeting the IL-12p35 subunit in mDC (33). As a consequence, supernatants from 495

cervical cancer-instructed mDC-fibroblasts co-cultures promoted the IL-17 production 496

in CD4+ memory T cells in an Il-23-dependent manner but failed to induce Th1 497

differentiation. Figure 7 summarizes our current concept of our findings. This is the 498




23

first report on a mechanism how the cervical cancer micromilieu can support Th17 499

expansion. 500

Th17 cells infiltrate precursor lesions and their number further increase during 501

cervical cancer progression (9,14). So far it was unclear how the Th17 expansion 502

occurs in cervical cancers. IL-23 has been shown to be necessary for Th17 503

expansion (20) and imbalances of DC-derived IL-23 and IL-12 expression in cancers 504

favored Th17 polarization (34). IL-23 exhibits pro-tumorigenic properties directly by 505

promoting tumor growth (35) or indirectly by supporting Th17 cells responses (20). 506

Th17 cells have a paradox role in different cancers. They mediate anti-tumor effects 507

by recruiting immune cells into tumors or stimulating effector CD8+ T cells (36,37) as 508

well as tumor-promoting responses driving proliferation, invasion, metastasis and 509

angiogenesis (38,39). Cervical cancer patients show increased serum levels of IL-23 510

which correlated with their Th17 numbers in the blood (22). In this study, our in situ 511

analysis showed CD83+ mDC co-expressing IL-23p19 in cervical cancer tissues. 512

Notably, the number of CD83+IL-23p19+ cells correlated with the numbers of Th17 513

cells. This correlation was stronger than the correlation between numbers of total IL-514

23p19+ or CD83- IL-23p19+ cells and Th17 cells, underlining the relevance of DC-515

derived IL-23. Furthermore patients with advanced tumor stages and lymph node 516

metastasis showed increased proportions of CD83+IL-23p19+ cells per total CD83+ 517

cells and CD4+IL-17+ cells per total CD4+ cells. Thus, the number of IL-23-expressing 518

mDC as well as IL-17-producing CD4+ T cells apparently increases during later 519

stages of carcinogenesis and is associated with the severity of the disease. 520

In our recent studies we observed that cervical cancer cells induce the chemokine 521

CCL20 in cervical fibroblasts supporting Th17 cell recruitment (9). Via expression of 522

pro-inflammatory cytokines and chemokines, such as IL-1 and CCL20 (9,40), 523




24

fibroblasts can promote inflammation during progression of different cancer types 524

(41). In peripheral tissues interactions between fibroblasts and DC can modulate DC 525

functions with respect to their ability to mature, migrate and trigger appropriate 526

adaptive immune responses (42). Depending on their anatomic region of origin 527

fibroblasts can transfer tissue-specific information to DC (43,44). Thus, DC co-528

cultured with renal fibroblasts showed decreased expression of CD80, CD83, CD86 529

and IL-12 but maintained normal levels of IL-23 and IL-27 (45). Skin-derived 530

fibroblasts did not influence maturation marker expression of mDC but support their 531

IL-23 expression and thereby Th17 responses (24) also after irradiation (46). In our 532

study, cervical fibroblasts did not interrupt efficient phenotypic maturation of mDC. 533

Furthermore, co-cultivation of cervical fibroblasts with mDC enhanced the IL-23 534

expression in normal as well as cervical cancer-instructed mDC. We identified IL-1 535

as the responsible factor produced by cervical cancer-instructed fibroblasts mediating 536

the increase of IL-23 expression in co-cultured mDC. Our data show that cervical 537

cancer cells instruct fibroblasts via IL-6/C/EBP to produce IL-1, thus engaging 538

same pathway previously described for the chemokine CCL20 (9). While fibroblasts-539

derived CCL20 supported the recruitment of Th17 cells (9), our neutralization 540

experiments clearly demonstrated that CCL20 was not involved in the expansion. 541

Rather, fibroblasts-derived IL-1 led to the IL-23-dependent expansion and enhanced 542

IL-17 production of Th17 cells. While the IL-1 expression declines in HPV-infected 543

epithelial cells during cervical carcinogenesis (47), we found stromal -SMA-positive 544

activated myofibroblasts in situ co-expressing IL-1 in cervical cancer biopsies. 545

Notably, in consecutive sections of the biopsy we also detected CD83+IL-23p19+ cells 546

in the same area of the tumor underlining our in vitro results. 547




25

IL-23 is a heterodimeric cytokine mainly expressed by DC containing the subunit IL-548

23p19 and shares the subunit IL-12p40 with IL-12 (20). Apart from IL-12p40 the 549

cytokine IL-12 consists of the subunit IL-12p35 which is regulated by the transcription 550

factor NF-B (28). We had previously described that recombinant IL-6 (48) or cervical 551

cancer cell-derived IL-6 suppresses the transcriptions factor NF-B in mDC (8). This 552

resulted in reduced expression of the chemokine receptor CCR7 in cancer-instructed 553

mDC (8) reducing their susceptibility for lymph node homing signals. Here we found 554

that cervical cancer cell-derived IL-6 dissociated the IL-12 from IL-23 expression in 555

cancer-instructed mDC by suppression of the IL-12p35. Cervical cancer cell-derived 556

IL-6 suppressed IL-12 production in mDC in the absence or presence of fibroblasts. 557

In contrast to p35, the IL-23p19 subunit is mainly regulated by MAPK p38 (49). IL-558

23p19 was not affected by IL-6, which is in line with our previous studies 559

demonstrating that recombinant IL-6 did not affect p38 activation in mDC (48). IL-6 is 560

highly upregulated during cervical carcinogenesis in situ and in vitro (12) and 561

correlated with poor prognosis of the patients (10,50). Cervical cancer cells display 562

only low responses to autocrine IL-6 due to low gp80 expression levels (12,51). 563

According to our previous studies (6,8,9,11), our results support the notion that IL-6 564

rather acts in a paracrine way to create a protumorigenic microenvironment. Thus, 565

cervical cancer cell-derived IL-6-instructed mDC-fibroblasts co-cultures mediated the 566

expansion of Th17 cells in an IL-23-dependent manner and reduced Th1 567

differentiation in vitro. By secretion of IL-6 and IL-1 cancer-instructed fibroblasts 568

supported the underlying diverse IL-23 and IL-12 production in co-cultured mDC. Our 569

study shows that IL-23 is an important mediator of Th17 expansion which may act in 570

concert with other factors of mDC-fibroblast supernatants. This differential 571

occurrence of Th17 and Th1 cells and their related cytokines is described for cervical 572




26

cancer patients. Patients exhibit elevated numbers of Th17 cells and increased IL-23- 573

and IL-17- but reduced IFN--levels in their serum (22) and decreased numbers of 574

HPV-specific Th1 cells (52). 575

Clinically most important, retrospective analysis from this study further supported the 576

in vivo relevance of our findings. We found that patients with increased numbers of 577

CD83+IL-23p19+ cells and Th17 cells developed lymph node metastasis and 578

recurrent cervical cancers. This may have major implications for personalization of 579

cervical cancer therapy suggesting the evaluation of the percentage of IL-23p19-580

expressing CD83+ cells per total tumor infiltrating CD83+ cells and proportion of IL-581

17-expressing CD4+ cells per total tumor infiltrating CD4+ cells as potential prognostic 582

markers or targets for immunotherapy. Inhibitors of IL-6 or of IL-6 signaling are 583

currently being evaluated for cancer treatment (53) that may target the IL-584

6/C/EBP/CCL20-dependent recruitment of tumor-promoting Th17 cells to cervical 585

cancer tissues (9). In mice CCR6-mediated T cell migration has been prevented 586

using an engineered CCL20 variant (54). Inhibitors of IL-6 may further target the 587

expression of IL-23 in DC and subsequent Th17 expansion. In contrast Th1 cells 588

expressing IFN- might be reconstituted, since IL-12, a major regulator of Th1 589

differentiation, is suppressed by IL-6. However, IL-6 inhibitors may prevent IL-590

6/STAT3-mediated sensitization of cervical cancer cells towards chemotherapeutic 591

drugs (51) and should therefore not be applied prior to irradiation or 592

chemoradiotherapy. Furthermore antibodies against IL-23p19, IL-17A or IL-17 593

receptor are currently being evaluated for treatment of psoriasis, inflammatory 594

diseases and different cancers (55) and should also be considered for cervical 595

cancer therapy. 596




27

In conclusion, our study identified a novel role of cervical cancer cell-derived IL-6 and 597

cervical fibroblasts with regard to Th17 cells in cervical cancers. Cervical fibroblasts 598

not only support Th17 infiltration via IL-6-mediated expression of the Th17-599

chemoatractant CCL20 (9), they also promote subsequent expansion of these pro-600

tumorigenic cells via secreted IL-6 and IL-1 by enhancing the expression of IL-23 in 601

cervical cancer-instructed mDC. The number of IL-23-expressing DC increases with 602

severity of disease and may explain the accumulation of Th17 cells in the stroma of 603

advanced cervical cancers, which may further support and maintain a chronic 604

protumorigenic inflammatory micromilieu, cervical cancer progression and may 605

influence cervical cancer therapy. 606

607

608

Acknowledgments 609

The authors thank Tanja Tänzer and Birgit Glombitza for excellent technical 610

assistance and all blood donors for providing their blood. This work was supported by 611

a grant of the Saarland University to B. Walch-Rückheim. B. Walch-Rückheim 612

received founding from the “Else Kröner-Fresenius-Stiftung” (2017-A64). 613

614

615

616

617




28

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52. de Vos van Steenwijk PJ, Piersma SJ, Welters MJ, van der Hulst JM, Fleuren G, 776

Hellebrekers BW, et al. Surgery followed by persistence of high-grade squamous 777

intraepithelial lesions is associated with the induction of a dysfunctional HPV16-778

specific T-cell response. Clin Cancer Res 2008;14:7188-95. 779

53. Taniguchi K, Karin M. IL-6 and related cytokines as the critical lynchpins between 780

inflammation and cancer. Semin Immunol 2014;26:54-74. 781

54. Getschman AE, Imai Y, Larsen O, Peterson FC, Wu X, Rosenkilde MM, et al. 782

Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in 783




33

an IL-23-dependent murine model. Proc Natl Acad Sci U S A 2017;114:12460-784

65. 785

55. Gaffen SL, Jain R, Garg AV, Cua DJ. The IL-23-IL-17 immune axis: from 786

mechanisms to therapeutic testing. Nat Rev Immunol 2014;14:585-600. 787

788

789




34

Figure legends: 790

Figure 1: CD83+IL-23p19+ cells infiltrate cervical SCCs and correlate with 791

numbers of Th17 cells, advanced FIGO stages, lymph node metastasis and 792

recurrent cervical cancers. 793

Sections of human SCCs were stained with a pan-cytokeratin Ab by IHC or costained 794

for CD83 (green) and IL-23p19 (red; A, upper panel), CD4 (green) and IL-17 (red; A, 795

lower panel) by immunofluorescence. (B) The number of CD83+IL-23p19+, (C) 796

number of total CD83+ and (D) number of CD83-IL-23p19+ cells was correlated with 797

the number of Th17 cells. (E) The percentage of CD83+IL-23p19+ cells/CD83 cells 798

correlated with the percentage of CD4+IL-17+ cells/CD4 cells. (F) Percentage of 799

CD83+IL-23p19+ cells/CD83 cells or (G) percentage of CD4+IL-17+ cells/CD4 cells 800

correlated with FIGO stages of the respective SCC, (H, I) with lymph node 801

metastasis or (J, K) recurrent cervical cancers. *0.05; **0.01. 802

803

Figure 2: Enhanced IL-23 but reduced IL-12 expression in cervical cancer-804

instructed mDC/NECF co-cultures. 805

(A) Time schedule of the experimental procedure. (B) Sections of human SCCs were 806

stained for pan-cytokeratin by IHC or costained for -SMA (red) and CD83 (green) by 807

immunofluorescence. Percentage of CD83+ cells with contact to -SMA+ cells was 808

evaluated. (C, E) Immature or TNF--stimulated mature normal or cervical cancer-809

instructed DC were cultured in medium alone (white and light grey bars) or used for 810

co-culture with NECF (white or grey bars) or NECF pre-stimulated with different CM 811

of cervical cancer cells (black bars). (D) Normal mDC were co-cultured with NECF 812

(grey bars) or fibroblasts from cervical cancer outgrowth cultures (black bars). Cell 813

free supernatants were analyzed for (C, D) IL-23 and (E) IL-12 expression. Shown 814




35

are the values SD averaged from n=3 experiments with 3 independent donors 815

performed in duplicates. **0.01; ***0.001. 816

817

Figure 3: Elevated levels of IL23p19 and IL-12p40 but reduced IL-12p35 in 818

cervical cancer-instructed mDC after co-cultivation with fibroblasts. 819

TNF--stimulated normal or cervical cancer-instructed DC were co-cultured with 820

NECF or NECF pre-stimulated with different CM of cervical cancer cells. Cells were 821

separated (NECF after co-culture: white bars; mDC after co-culture with NECF: grey 822

bars; mDC after co-culture with pre-stimulated NECF: black bars) and levels of (A) IL-823

23p19-, (B) IL-12p40- and (C) IL-12p35-specific mRNA were quantified using real-824

time PCR in relation to -Actin. mRNA levels of normal mDC were set at 1. Shown 825

are the values SD averaged from n=3 experiments from 3 independent donors 826

performed in duplicates. *0.05; **0.01; ***0.001. 827

828

Figure 4: Cervical cancer cells suppressed the IL-12p35 subunit in mDC via IL-829

6. 830

(A) DC were generated in the presence of medium or increasing doses of rhIL-6. 831

TNF--stimulated mDC were cultured alone or co-cultured with NECF. Cell free 832

supernatants were analyzed for IL-12 expression. (B, C, D) Cells were generated and 833

stimulated as in (A). Cells were separated and levels of (B) IL-12p35-, (C) IL-12p40- 834

and (D) IL-23p19-specific mRNA were quantified using real-time PCR in relation to -835

Actin. MRNA levels of normal iDC were set at 1. (E) Supernatants of different cervical 836

cancer cells were analyzed for IL-6 expression. (F, G) TNF--stimulated normal or 837

cervical cancer-instructed DC were co-cultured with NECF. For IL-6 neutralization 838

conditioned media were preincubated with anti-IL-6 or isotype control antibodies 839




36

during generation of cervical cancer-instructed mDC. Cell free supernatants were 840

analyzed for (F) IL-12 and (G) IL-23 expression. Shown are the mean values SD 841

averaged from n=3 experiments performed in triplicates. **0.01; ***0.001; n.s., non 842

significant. 843

844

Figure 5: Cervical cancer cells induce IL-1 expression in fibroblasts in an IL-845

6/C/EBP-dependent manner increasing IL-23 expression in co-cultured mDC. 846

(A) Time schedule of the experimental procedures in B-G. (B, C) NECF were 847

stimulated with medium or conditioned media from NECK or different cervical cancer 848

cells. (B) IL-1 mRNA expression levels were quantified by real-time PCR in relation 849

to RPL13A. (C) Supernatants were collected and analyzed for IL-1 expression. (D) 850

NECF were stimulated with medium or conditioned media of cervical cancer cells. 851

For IL-6 neutralization, conditioned media were preincubated with anti-IL-6 or 852

isotype-control antibodies for 2 h. Supernatants were analyzed for IL-1 expression. 853

(E, F) NECF were transfected with 10 pmol of human C/EBP-specific siRNA or 854

control siRNA/1.5 x 105 cells. (E) After 48 h whole cell extracts were analyzed for 855

C/EBP expression by Western blot analysis; diagram shows quantification of n=3 856

experiments. (F) Cells were stimulated with medium or conditioned medium from 857

cervical cancer cells and supernatants were analyzed for IL-1 expression. (G). 858

NECF were pre-stimulated with conditioned media of SW756 or SiHa cells. 859

Stimulated fibroblasts were incubated with anti-IL-1 or isotype control antibodies 860

and co-cultured with TNF--stimulated normal or cervical cancer-instructed mDC. 861

Cell free supernatants were analyzed for IL-23 expression. (H, I) Supernatants of 862

NECF and fibroblasts from cervical cancer outgrowth cultures from three different 863

donors were analyzed for IL-6 (H) and IL-1 (I) expression. (J) TNF--stimulated 864




37

fibroblasts- or cervical cancer-instructed DC were co-cultured with fibroblasts from 865

cervical cancer outgrowth cultures. For IL-6 neutralization conditioned media were 866

preincubated with anti-IL-6 or isotype control antibodies during generation of 867

fibroblasts- or cervical cancer-instructed mDC or before co-cultures. (K) Fibroblasts 868

from cervical cancer outgrowth cultures were incubated with anti-IL-1 or isotype 869

control antibodies and co-cultured with TNF--stimulated fibroblasts- or cervical 870

cancer-instructed mDC. (J, K) Cell free supernatants were analyzed for IL-23 871

expression. Shown are the values SD averaged from n=3 experiments performed 872

in triplicates. **0.01; ***0.001. (L) Sections of human SCCs were stained for pan-873

cytokeratin by IHC and costained for -SMA (green) and IL-1 (red; upper panel), 874

CD83 (green) and IL-23p19 (red; lower panel) by IF. 875

876

Figure 6: Supernatants of cervical cancer-instructed mDC-NECF co-cultures 877

support IL-17 production of memory CD4+ T cells via IL-23. 878

(A) Scheme of the experimental procedures in B-H. CD4+ memory T cells stimulated 879

with CD2/CD3/CD28 beads were incubated with medium, rhIL-23 or supernatants of 880

mDC-fibroblasts co-cultures generated with normal or cervical cancer-instructed 881

mDC and NECF or pre-stimulated NECF. (B, C, D) On day 6, cells were analyzed for 882

CD4, IL-17 and IFN- expression using flow cytometry and (D) Trucount™ Absolute 883

Counting Tubes. Shown is one representative experiment (B); diagram shows 884

quantification of n=3 experiments (C, D). (E) Cell free supernatants were analyzed for 885

IL-17 expression and normalized to 1000 Th17 cells. (F, G, H) CD4+ memory T cells 886

were stimulated and incubated as described above. For IL-23 neutralization, 887

supernatants of mDC/NECF co-cultures were pre-incubated with neutralizing IL-23 888

antibodies or isotype control antibodies. On day 6, (F, G) cells were analyzed for 889




38

CD4 and IL-17 expression, numbers of Th17 cells were determined (G) or (F) cell 890

free supernatants were analyzed for IL-17 expression. (I, J, K) Fibroblasts from 891

cervical cancer outgrowth cultures were incubated with anti-IL-1 or isotype control 892

antibodies and co-cultured with TNF--stimulated fibroblasts- or cervical cancer-893

instructed DC. Supernatants were harvested and used for stimulation of CD4+ 894

memory T cells. On day 6, (I, J) cells were analyzed for CD4 and IL-17 expression, 895

numbers of Th17 cells were determined (J) or (K) cell free supernatants were 896

analyzed for IL-17 expression. Shown are the values SD averaged from n=3 897

experiments with 3 independent donors performed in triplicates.. *0.05; **0.01; 898

***0.001. 899

900

Figure 7: Schematic presentation of the influence of cervical cancer cell- and 901

fibroblast-derived IL-6 on the differential IL-23 and IL-12 regulation in DC, 902

C/EBP-dependent IL-1 expression by stromal fibroblasts and subsequent 903

Th17 expansion. 904

905

906




Published OnlineFirst January 29, 2019.Cancer Res Barbara Walch-Rückheim, Russalina Ströder, Laura Theobald, et al. cellsexpression in dendritic cells to support expansion of Th17 Cervical cancer-instructed stromal fibroblasts enhance IL-23

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