**Author details**

Th Helper T cell

TLR Toll-like receptor Treg Regulatory T cell

**A.1. Experimental procedures**

**A. Appendices and nomenclature**

112 Cervical Cancer - Screening, Treatment and Prevention - Universal Protocols for Ultimate Control

Biotec) and analyzed using MACSQuantify software.

was considered to be statistically significant at p < 0.05.

reaction mix (Evrogen, Russia).

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.

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

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

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 Olga Kurmyshkina<sup>1</sup> , Pavel Kovchur<sup>2</sup> , Ludmila Schegoleva3 and Tatyana Volkova4,5\*

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

1 Laboratory of Molecular Genetics of Innate Immunity, Institute of High-Tech Biomedicine, Petrozavodsk State University, Petrozavodsk, Russian Federation

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

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

4 Department of Biomedical Chemistry, Immunology and Laboratory Diagnostics, Institute of Medicine, Petrozavodsk State University, Petrozavodsk, Russian Federation

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