**A. Appendices and nomenclature**

#### **A.1. Experimental procedures**

Patients and specimens: Samples of peripheral blood and epithelial tissue were obtained from 55 patients who were diagnosed with CIN2-3 (including cancer *in situ*) or microinvasive carcinoma (FIGO stage IA1) and underwent surgery in Oncological Dispensary of the Republic of Karelia. CIN and cervical cancer diagnosis was based on comprehensive physical examination, extended colposcopy findings, cytology, and histopathology tests, in full compliance with the approved standards for the diagnosis and treatment of patients with gynecological malignancies. All women engaged in this this study were informed and gave voluntary written consent. The research was approved by the Committee on Medical Ethics of Petrozavodsk State University and the Ministry of Healthcare and Social Development of the Republic of Karelia, and was done in accordance with the Declaration of Helsinki and good clinical practice guidelines. All women from patient group were positive for oncogenic HPV types (with the prevalence of HPV16 > 80%). Thirty healthy non-pregnant women without cervical abnormalities and HPV-infection at the time of blood sampling served as normal controls. Venous blood was collected immediately before the surgery or any other treatment and immediately processed for multicolor flow cytometry. Tissue samples were submerged in RNA stabilizing reagent right after excision and stored at −80.

**Author details**

Olga Kurmyshkina<sup>1</sup>

Federation

Federation

**References**

, Pavel Kovchur<sup>2</sup>

Petrozavodsk State University, Petrozavodsk, Russian Federation

\*Address all correspondence to: volkovato@yandex.ru

, Ludmila Schegoleva3

Immune Regulatory Network in Cervical Cancer Development: The Expanding Role of Innate...

1 Laboratory of Molecular Genetics of Innate Immunity, Institute of High-Tech Biomedicine,

2 Department of Hospital Surgery, ENT Diseases, Ophthalmology, Dentistry, Oncology, Urology, Institute of Medicine, Petrozavodsk State University, Petrozavodsk, Russian

3 Department of Applied Mathematics and Cybernetics, Institute of Mathematics and Information Technologies, Petrozavodsk State University, Petrozavodsk, Russian

of Medicine, Petrozavodsk State University, Petrozavodsk, Russian Federation

Open Biology. 2017;**7**(4):170006. DOI: 10.1098/rsob.170006

therapy. Virus. 2017;**9**(9):E254. DOI: 10.3390/v9090254

Pathology. 2016;**238**(2):166-179. DOI: 10.1002/path.4656

Virus. 2017;**9**(10):E293. DOI: 10.3390/v9100293

82. DOI: 10.1177/2051013617717914

fimmu.2017.00689

4 Department of Biomedical Chemistry, Immunology and Laboratory Diagnostics, Institute

5 Institute of High-Tech Biomedicine, Petrozavodsk State University, Petrozavodsk, Russia

[1] Calì B, Molon B, Viola A. Tuning cancer fate: The unremitting role of host immunity.

[2] Smola S. Immunopathogenesis of HPV-associated cancers and prospects for immuno-

[3] Alizon S, Murall CL, Bravo IG. Why human papillomavirus acute infections matter.

[4] Seelige R, Searles S, Bui JD. Innate sensing of cancer's non-immunologic hallmarks.

[5] Doorbar J. Model systems of human papillomavirus-associated disease. The Journal of

[6] Smola S, Trimble C, Stern PL. Human papillomavirus-driven immune deviation: Challenge and novel opportunity for immunotherapy. Therapeutic Advances in Vaccines. 2017;**5**(3):69-

[7] Qin Y, Ekmekcioglu S, Forget MA, Szekvolgyi L, Hwu P, Grimm EA, Jazaeri AA, Roszik J. Cervical cancer neoantigen landscape and immune activity is associated with human papillomavirus master regulators. Frontiers in Immunology. 2017;**8**:689. DOI: 10.3389/

Current Opinion in Immunology. 2017;**50**:1-8. DOI: 10.1016/j.coi.2017.09.005

and Tatyana Volkova4,5\*

http://dx.doi.org/10.5772/intechopen.72518

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Flow cytometry: The following fluorophore-conjugated monoclonal antibodies were used: CD3-APC (Clone: UCHT1), CD4-FITC (Clone: MT310), CD8-FITC (Clone: DK25), CD16-FITC (Clone: DJ130c), CD56-RPE (Clone: C5.9) (Dako, Austria), CD25-APC (Clone: 4E3), CD45- VioBlue (Clone: 5B1), CD127-RPE (Clone: MB15-18C9), FoxP3-RPE (Clone: 3G3) (Miltenyi Biotec, Germany), STING/TMEM173-RPE (Clone: 723505, R&D Systems, USA). For blocking of non-specific antibody binding, FcR Blocking Reagent (Miltenyi Biotec) was used. For intracellular detection, cells were fixed and permeabilized using "FoxP3 Staining Buffer Set" (Miltenyi Biotec). Cells were acquired on a MACSQuant Analyzer flow cytometer (Miltenyi Biotec) and analyzed using MACSQuantify software.

Real-time PCR: Total RNA was extracted from tissue samples or ficoll-isolated PBMC with Trizol Reagent (Invitrogen). cDNA was synthesized from DNAse I-treated RNA (1 mg RNA per 1 reaction volume) using ProtoScript II (New England BioLabs, UK) or RevertAid First Strand cDNA Synthesis Kit (Fermentas, ThermoScientific, USA). Amplification was performed in StepOnePlus thermal cycler (Applied Biosystems, USA) using qPCRmix-HS-SYBR+HighROX reaction mix (Evrogen, Russia).

Statistical analysis: Data analysis was performed using R software. Mann-Whitney U-test was used to evaluate the differences between the patient and the control groups; the difference was considered to be statistically significant at p < 0.05.
