**2. Intrinsic molecular mechanisms bridging antiviral and antitumor immune responses in cervical cancer**

#### **2.1. The role of nucleic acid-sensing pattern-recognition receptors (PRRs) and related signaling pathways in controlling cervical cancer development: current concepts**

increasing the risk of malignant transformation. In the later stages of carcinogenesis, in contrast to the stage of productive infection, HPV-transformed cells reprogram their environment in such a way that they gain the ability to recruit different populations of immune cells and to initiate chronic stromal inflammation, which contributes to further progression of precursor lesions into invasive cancer, facilitates tumor growth and metastastic spreading, and simulta-

**Figure 1.** Scheme illustrating general relations between the key levels of immune response to cervical cancer that are

As a result of the fact that cervical cancer development is characterized by high genomic instability, the accumulated somatic mutations generate the enormous variety of neoantigens, which, together with the HPV-antigens, represent the potential targets for the T cell-mediated adaptive (TCR-restricted) response [6]. The range and immunogenicity (the ability to be presented to cytotoxic and helper T cells) of these antigens have been proved in high-throughput studies using integrated approaches to genome/transcriptome sequencing data analysis (see, for example, [7]). At the same time, the study by Qin et al. shows that increased mutation burden and neoantigen load correlates with HPV-dependent activation of master regulator genes that abrogate antitumor immune responses these neoantigens could cause by mobilizing immune regulatory, suppressive mechanisms. This again proves the rationale of studying the innate and innate-like lymphocytes, regulatory T/B lymphocytes, cells of myeloid lineage, as well as the mechanisms of antigen-independent innate immune response (including those involving DNA sensors) and the processes of immune regulation at different stages of cervical neoplasia development. In present chapter, the results of studies on these specific cell populations, mechanisms and processes published in last 2–3 years are described, with simultaneous discussion of our own experimental data on this problem, obtained from the patients with the diagnosis of pre- and microinvasive cervical cancer. Since a large number of constantly updated reviews are available on the issue of molecular strategies used by HPV to avoid immune response or other so-called cell restriction factors (see, for example, [8]), this question is not presented in the Chapter. In addition, we do

neously promotes exhaustion of effector immune cells populations [2].

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

addressed in the chapter.

To respond to ectopically localized nucleic acids of exogenous (infectious) or endogenous (tumor cell- or stressed cell-derived) origin, cells are "armed" with a set of nucleic acid-sensing pattern recognition receptors (PRRs). Members of this essential group of PRRs are expressed in cells of both immune (lymphoid/myeloid) and non-immune (for example, epithelial) origin and can recognize various forms of nucleic acids (single- and double-stranded DNA or RNA, DNA-RNA-heteroduplexes, CpG-islets, as well as specific chemical modifications or structures, typical for viral DNA/RNA, and messenger cyclic nucleotides) in different cellular compartments (cytosol, endosomes/phagosomes, and even in the nucleus). These include some representatives of Toll-like receptor family (TLRs: 3, 7, 8, 9), Absent in Melanoma 2 family, (AIM2, IFI16), RIG-I-like receptors (RLRs: RIG-I and MDA5), and other members of the DExD/H helicase family, as well as a "signaling pair" of cyclic GMP-AMP synthase (cGAS)—Stimulator of Interferon Genes (STING). In spite of the fact that these receptors/sensors activate different signaling pathways, they all eventually lead to the activation of transcription factors such as Interferon Regulatory Factors (IRFs) or Nuclear Factor kappa B (NF-kB), which are responsible for the production of type I interferons (IFN-I) or proinflammatory cytokines, respectively [12].

Among the listed molecular sensors, the STING protein is recognized as a signaling hub (**Figure 2A**): it can receive and redistribute signals coming from different upstream molecular partners, although the most well studied and, perhaps, most important for mammalian cells, is the cGAS-STING signaling axis [13]. Binding of cGAS with cytosolic DNA results in the synthesis of secondary messenger—cyclic dinucleotide cGAMP—a natural STING ligand; following interaction with cGAMP, STING (an endoplasmic reticulum membrane-resident protein) initiates assembly of a multiprotein complex (i.e., signaling platform) and, through activation of IRF3 transcription factor, triggers expression of a large number of genes, including IFN-I genes and IFN-stimulated genes (ISG). Moreover, the new data from high-throughput transcriptome analysis showed that depending on the cell type, STING can alter the expression of not only the immune responseassociated genes, but also many other genes that govern crucial cellular processes (proliferation, apoptosis, and stress response) [14–16]. The existence of alternative pathways that lead to STING activation (possibly ligand/agonist-independent) is also assumed, although the mechanisms have not yet been sufficiently described [17]. The key role of STING in antiviral innate immune defense has been confirmed by numerous studies, and it is not surprising, therefore, that different groups of viruses have evolved a variety of strategies to avoid/inhibit STING-dependent response, and oncoviruses are no exception: for most of tumor-associated mammalian viruses, STING, and other components of the STING-dependent signaling pathway were found to be specifically targeted by viral oncoproteins (in our previous paper, we summarized known mechanisms that are used by the oncoviruses, in particular, HPV, to evade STING-mediated recognition [18]).

> As mentioned before, STING-mediated signaling has been most thoroughly investigated in macrophages and dendritic cells while its role in other cell populations, specifically non-myeloid cells, is not fully understood. In this respect, recently published findings from *in vitro* and *in vivo* experiments (carried out using genetically engineered mice and STING ligands/agonists) demonstrating the functionality of canonical STING-dependent signaling in T cells [14–16] are of high importance. Surprisingly, besides activation of IFN-I response these experiments revealed T cell-specific ability of STING to modulate (inhibit) TCR-stimulated expansion and to induce cell death (through IRF3- and p53-dependent pathway), which is the fundamental difference from macrophages, in which stimulation of STING never leads to activation of death-associated genes [14–16]. The T cell-specific effect is extremely important for the prediction of therapeutic effect of STING agonists, which are currently undergoing extensive clinical trials as adjuvants in chemo-and immunotherapy of different types of cancer; however, in the case of cervical cancer the specificity of STING expression changes has not been investigated so far. At the same time, HPV-associated cervical cancer, in our opinion, can be used as a model object to study either cell type-specific or stage-specific involvement of STING in the innate/adaptive immune function-

> **Figure 2.** (A) Activating signals from various sources converge on STING to initiate cell type-specific innate response to cytosolic DNA. (B) In HPV-induced neoplastic lesions, STING can receive activating signals both from invading HPV-

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**2.2. Altered patterns of STING expression indicate its putative role in cervical cancer**

Based on the above facts, a study of the expression profile of STING (at mRNA/protein level) in tissue samples as well as in the major populations of peripheral blood T lymphocytes obtained from patients with preinvasive and microinvasive cervical cancer compared to healthy women (control group) has been started by our research team. We also took into account that: (1) increased expression of markers of apoptosis can be observed in circulating T lymphocytes in patients with early (pre-clinical) stages of cervical cancer [23]; (2) patients with early-stage cancer or precursor lesions display a variety of systemic alterations in the immune system including altered phenotype/activity and frequencies of different T cell populations, as evidenced by the large number of data (including those described below); (3) HPV-DNA (and possibly tumor DNA) circulates in the body and thus can be detected in various tissues and lymphoid organs long before the first detectable signs of metastases [24], whereby it potentially exerts a

ing at local and, most importantly, systemic level.

DNA and mislocalized self-DNA.

systemic effect on the activity of the STING.

The involvement of STING in regulation of the relationship between the tumor and the immune system (both innate and adaptive branches) mediated through the recognition of tumor DNA has been experimentally corroborated, although there are still many unresolved contradictions regarding its precise role in carcinogenesis: in different mouse tumor models, stimulation of expression, and/or activity of STING resulted in either restriction of tumor growth or tumor progression. What reasons could underlie these contradictions? On one hand, the STING-induced production of type I IFNs and activation of inflammatory reactions are obviously indispensable for the proper functioning of antigen-presenting cells (APC) and for further induction of adaptive antitumor response (discussed in [13, 19, 20]). On the other hand, the increased activity of STING leads to chronic inflammation within the locus of neoplasia which is a driving force of immunosuppression and tumorigenesis. In addition, there is still no clear understanding of exactly which cells within a tumor are responsible for STING-dependent recognition of tumor DNA. A previously proposed model, according to which it is phagocytizing cells (primarily dendritic cells and macrophages) that can engulf tumor DNA from dead/apoptotic tumor cells and activate the STING-signaling pathway, causes many doubts as it is not clear how endosomal/phagosomal DNA can reach cytosolic cGAS.Another model has been recently proposed, whereby the primary recognition of tumor DNA and synthesis of cGAMP occurs in tumor cells themselves because of the "leakage" of nuclear DNA into the cytosol (as a result of genomic instability, DNA damage, increased proliferation rates); cGAMP can diffuse to neighboring cells, including immune ones presumably during the formation of immunological synapse—which are more efficient IFN-I producers and thus are able to promote recruitment of dendritic cells (DCs) and effector T cells [21]. APCs are widely recognized as such efficient producers, but other types of cells, for instance, lymphoid cells, can also be the candidates, considering that the level of STING mRNA/protein expression in lymphocytes was shown to be significantly higher than in macrophages [14, 16]. This model assumes that the initial stages of carcinogenesis are accompanied by an increased expression/activity of cGAS-STING, but as the tumor progresses, a disruption of cGAS-STING signaling—as a way to counterattack anti-tumor immunity—can occur. However, in virus-associated cancers, including cervical cancer, where STING activity can potentially be modulated by virus-derived and tumor-derived DNA, there may be the opposite sequence of events: in the initial phase of the establishment of a chronic infection, viral oncoproteins inhibit cGAS-STING pathway in infected cells (**Figure 2B**) and then, after undergoing malignant transformation, tumor cells gain the ability to support up-regulated state of cGAS-STING signaling in order to generate inflammatory immunosupressive microenvironment. Immunohistochemical study of HPV-infected cervical epithelium and low-grade cervical lesions indeed showed reduced expression of STING in relation to normal epithelium [22], but what changes are characteristic of high grade lesions and cervical cancer are as yet unknown.

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activation (possibly ligand/agonist-independent) is also assumed, although the mechanisms have not yet been sufficiently described [17]. The key role of STING in antiviral innate immune defense has been confirmed by numerous studies, and it is not surprising, therefore, that different groups of viruses have evolved a variety of strategies to avoid/inhibit STING-dependent response, and oncoviruses are no exception: for most of tumor-associated mammalian viruses, STING, and other components of the STING-dependent signaling pathway were found to be specifically targeted by viral oncoproteins (in our previous paper, we summarized known mechanisms that are used by

The involvement of STING in regulation of the relationship between the tumor and the immune system (both innate and adaptive branches) mediated through the recognition of tumor DNA has been experimentally corroborated, although there are still many unresolved contradictions regarding its precise role in carcinogenesis: in different mouse tumor models, stimulation of expression, and/or activity of STING resulted in either restriction of tumor growth or tumor progression. What reasons could underlie these contradictions? On one hand, the STING-induced production of type I IFNs and activation of inflammatory reactions are obviously indispensable for the proper functioning of antigen-presenting cells (APC) and for further induction of adaptive antitumor response (discussed in [13, 19, 20]). On the other hand, the increased activity of STING leads to chronic inflammation within the locus of neoplasia which is a driving force of immunosuppression and tumorigenesis. In addition, there is still no clear understanding of exactly which cells within a tumor are responsible for STING-dependent recognition of tumor DNA. A previously proposed model, according to which it is phagocytizing cells (primarily dendritic cells and macrophages) that can engulf tumor DNA from dead/apoptotic tumor cells and activate the STING-signaling pathway, causes many doubts as it is not clear how endosomal/phagosomal DNA can reach cytosolic cGAS.Another model has been recently proposed, whereby the primary recognition of tumor DNA and synthesis of cGAMP occurs in tumor cells themselves because of the "leakage" of nuclear DNA into the cytosol (as a result of genomic instability, DNA damage, increased proliferation rates); cGAMP can diffuse to neighboring cells, including immune ones presumably during the formation of immunological synapse—which are more efficient IFN-I producers and thus are able to promote recruitment of dendritic cells (DCs) and effector T cells [21]. APCs are widely recognized as such efficient producers, but other types of cells, for instance, lymphoid cells, can also be the candidates, considering that the level of STING mRNA/protein expression in lymphocytes was shown to be significantly higher than in macrophages [14, 16]. This model assumes that the initial stages of carcinogenesis are accompanied by an increased expression/activity of cGAS-STING, but as the tumor progresses, a disruption of cGAS-STING signaling—as a way to counterattack anti-tumor immunity—can occur. However, in virus-associated cancers, including cervical cancer, where STING activity can potentially be modulated by virus-derived and tumor-derived DNA, there may be the opposite sequence of events: in the initial phase of the establishment of a chronic infection, viral oncoproteins inhibit cGAS-STING pathway in infected cells (**Figure 2B**) and then, after undergoing malignant transformation, tumor cells gain the ability to support up-regulated state of cGAS-STING signaling in order to generate inflammatory immunosupressive microenvironment. Immunohistochemical study of HPV-infected cervical epithelium and low-grade cervical lesions indeed showed reduced expression of STING in relation to normal epithelium [22], but what changes are characteristic of high

the oncoviruses, in particular, HPV, to evade STING-mediated recognition [18]).

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

grade lesions and cervical cancer are as yet unknown.

**Figure 2.** (A) Activating signals from various sources converge on STING to initiate cell type-specific innate response to cytosolic DNA. (B) In HPV-induced neoplastic lesions, STING can receive activating signals both from invading HPV-DNA and mislocalized self-DNA.

As mentioned before, STING-mediated signaling has been most thoroughly investigated in macrophages and dendritic cells while its role in other cell populations, specifically non-myeloid cells, is not fully understood. In this respect, recently published findings from *in vitro* and *in vivo* experiments (carried out using genetically engineered mice and STING ligands/agonists) demonstrating the functionality of canonical STING-dependent signaling in T cells [14–16] are of high importance. Surprisingly, besides activation of IFN-I response these experiments revealed T cell-specific ability of STING to modulate (inhibit) TCR-stimulated expansion and to induce cell death (through IRF3- and p53-dependent pathway), which is the fundamental difference from macrophages, in which stimulation of STING never leads to activation of death-associated genes [14–16]. The T cell-specific effect is extremely important for the prediction of therapeutic effect of STING agonists, which are currently undergoing extensive clinical trials as adjuvants in chemo-and immunotherapy of different types of cancer; however, in the case of cervical cancer the specificity of STING expression changes has not been investigated so far. At the same time, HPV-associated cervical cancer, in our opinion, can be used as a model object to study either cell type-specific or stage-specific involvement of STING in the innate/adaptive immune functioning at local and, most importantly, systemic level.

#### **2.2. Altered patterns of STING expression indicate its putative role in cervical cancer**

Based on the above facts, a study of the expression profile of STING (at mRNA/protein level) in tissue samples as well as in the major populations of peripheral blood T lymphocytes obtained from patients with preinvasive and microinvasive cervical cancer compared to healthy women (control group) has been started by our research team. We also took into account that: (1) increased expression of markers of apoptosis can be observed in circulating T lymphocytes in patients with early (pre-clinical) stages of cervical cancer [23]; (2) patients with early-stage cancer or precursor lesions display a variety of systemic alterations in the immune system including altered phenotype/activity and frequencies of different T cell populations, as evidenced by the large number of data (including those described below); (3) HPV-DNA (and possibly tumor DNA) circulates in the body and thus can be detected in various tissues and lymphoid organs long before the first detectable signs of metastases [24], whereby it potentially exerts a systemic effect on the activity of the STING.

*2.2.1. STING protein levels in different subsets of circulating lymphocytes from early-stage cervical cancer patients*

Intracellular STING level was measured in circulating CD4 and CD8 T cells, as well as in CD4CD25 subset (**Figure 3**) by flow cytometry using anti-human STING monoclonal antibody (MAb; clone 723505). Since the majority of lymphocyte population were stained positively for STING (which is in compliance with previously reported data showing that STING is robustly expressed in lymphoid tissue, specifically in T cells [14]), making the percentage values less informative, the level of STING protein was expressed as relative Mean Fluorescence Intensity value (ΔMFI) normalized to MFI of isotype control (IgG) with correction for autofluorescence of corresponding T cell subsets (Fluorescence Minus One, or FMO, control) (**Figure 3**).

As we did not find published works reporting on the level of STING in peripheral blood lymphocytes analyzed by means of immunofluorescence techniques, we first compared different commercially available kits for intracellular protein staining. The results of intracellular STING evaluation in peripheral blood T cells appeared to be sensitive to the permeabilizing ability of a fixation/permeabilization buffer set used, specifically: when kits designed for staining of intracellular proteins (such as cytokines) were applied, the level of anti-STING MAb binding did not differ from isotype control (**Figure 4A**); whereas the use of a reagent kit intended for intracellular detection of antigens such as nuclear transcription factors resulted in significant anti-STING MAb binding compared to isotype control (**Figure 4B**). This might be due to specific localization of STING and availability of its epitopes: homodimeric STING resides in the ER membrane and upon activation may form aggregates and translocate to Golgi and perinuclear space [25] (according to the manufacturer, the immunogen aa215-379 for the clone 723505 of anti-STING MAb corresponds to the C-terminal cytoplasmic domain of human STING).

In early-stage cervical cancer patients (with carcinoma *in situ* or microinvasive carcinoma), the level of STING protein showed a decreasing trend in both CD4 and CD8 T subsets compared to healthy controls with this decrease being more pronounced in CD8 T lymphocytes (**Figure 5A**). No significant change was observed for CD4CD25 subpopulation. A notable increase in ΔMFI (CD4CD25)/ΔMFI (CD3CD8) ratio was revealed for circulating T cells from cancer patients (**Figure 5B**), implying that STING expression became more pronounced in CD4CD25 lymphocytes in relation to CD3CD8 subset. At the same time, the difference between STING levels in CD3CD4 and CD3CD8 cells from both controls and cancer patients was less significant; ΔMFI(CD3CD4)/ΔMFI (CD3CD8) ratios were close to 1 in all groups studied suggesting that the expression of STING is associated with both CD4 and CD8 T cell subsets. These results are, in a certain sense, in consistence with data reported previously by others for mouse models [14]. The percentage of STING-positive cells in total population of circulating lymphocytes from cervical cancer patients was on average lower than that in the control group, although this difference was not statistically significant (p > 0.05, U-test; **Figure 6**). When analyzing CD3 T cells, the same trend could be observed (while the total frequencies of T cells did not differ

**Figure 4.** Representative examples of STING staining carried out using (A) a reagent buffer set for staining cytosolic proteins (e.g., cytokines), or (B) a reagent kit with stronger permeabilizing capacity for intracellular/nuclear protein

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*2.2.2. STING mRNA expression in peripheral blood mononuclear cells (PBMC) and neoplastic* 

At the mRNA level, STING expression was analyzed in ficoll-isolated PBMC using semi-qPCR (RPLP0 and PGK1 genes were used as endogenous controls [26]): similar to flow cytometry results, in PBMC from patients with preinvasive/microinvasive cancer (stage 0-IA), STING-mRNA

between patients and controls).

*tissue samples*

staining.

**Figure 3.** T cell gating and evaluation of STING protein level.

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*2.2.1. STING protein levels in different subsets of circulating lymphocytes from early-stage* 

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

of corresponding T cell subsets (Fluorescence Minus One, or FMO, control) (**Figure 3**).

As we did not find published works reporting on the level of STING in peripheral blood lymphocytes analyzed by means of immunofluorescence techniques, we first compared different commercially available kits for intracellular protein staining. The results of intracellular STING evaluation in peripheral blood T cells appeared to be sensitive to the permeabilizing ability of a fixation/permeabilization buffer set used, specifically: when kits designed for staining of intracellular proteins (such as cytokines) were applied, the level of anti-STING MAb binding did not differ from isotype control (**Figure 4A**); whereas the use of a reagent kit intended for intracellular detection of antigens such as nuclear transcription factors resulted in significant anti-STING MAb binding compared to isotype control (**Figure 4B**). This might be due to specific localization of STING and availability of its epitopes: homodimeric STING resides in the ER membrane and upon activation may form aggregates and translocate to Golgi and perinuclear space [25] (according to the manufacturer, the immunogen aa215-379 for the clone 723505 of anti-STING MAb corresponds to the C-terminal cytoplasmic domain of human STING).

In early-stage cervical cancer patients (with carcinoma *in situ* or microinvasive carcinoma), the level of STING protein showed a decreasing trend in both CD4 and CD8 T subsets compared to

**Figure 3.** T cell gating and evaluation of STING protein level.

Intracellular STING level was measured in circulating CD4 and CD8 T cells, as well as in CD4CD25 subset (**Figure 3**) by flow cytometry using anti-human STING monoclonal antibody (MAb; clone 723505). Since the majority of lymphocyte population were stained positively for STING (which is in compliance with previously reported data showing that STING is robustly expressed in lymphoid tissue, specifically in T cells [14]), making the percentage values less informative, the level of STING protein was expressed as relative Mean Fluorescence Intensity value (ΔMFI) normalized to MFI of isotype control (IgG) with correction for autofluorescence

*cervical cancer patients*

**Figure 4.** Representative examples of STING staining carried out using (A) a reagent buffer set for staining cytosolic proteins (e.g., cytokines), or (B) a reagent kit with stronger permeabilizing capacity for intracellular/nuclear protein staining.

healthy controls with this decrease being more pronounced in CD8 T lymphocytes (**Figure 5A**). No significant change was observed for CD4CD25 subpopulation. A notable increase in ΔMFI (CD4CD25)/ΔMFI (CD3CD8) ratio was revealed for circulating T cells from cancer patients (**Figure 5B**), implying that STING expression became more pronounced in CD4CD25 lymphocytes in relation to CD3CD8 subset. At the same time, the difference between STING levels in CD3CD4 and CD3CD8 cells from both controls and cancer patients was less significant; ΔMFI(CD3CD4)/ΔMFI (CD3CD8) ratios were close to 1 in all groups studied suggesting that the expression of STING is associated with both CD4 and CD8 T cell subsets. These results are, in a certain sense, in consistence with data reported previously by others for mouse models [14].

The percentage of STING-positive cells in total population of circulating lymphocytes from cervical cancer patients was on average lower than that in the control group, although this difference was not statistically significant (p > 0.05, U-test; **Figure 6**). When analyzing CD3 T cells, the same trend could be observed (while the total frequencies of T cells did not differ between patients and controls).

#### *2.2.2. STING mRNA expression in peripheral blood mononuclear cells (PBMC) and neoplastic tissue samples*

At the mRNA level, STING expression was analyzed in ficoll-isolated PBMC using semi-qPCR (RPLP0 and PGK1 genes were used as endogenous controls [26]): similar to flow cytometry results, in PBMC from patients with preinvasive/microinvasive cancer (stage 0-IA), STING-mRNA

**Figure 5.** (A) The change of STING protein levels in major subsets of peripheral blood T cells from patients with precancerous cervical lesions or cancer (stage 0-IA, n = 20) relative to the control group of healthy donors (n = 15). (B) The ratio of the relative STING expression level in different T cell populations. Mean ± SEM values are displayed; p-value was assessed by U-test.

in each patient group showed elevated STING expression as compared with normal noninfected epithelium (though the group mean values did not differ statistically). Up-regulation of STING at early stages of cervical carcinogenesis is consistent with some previous reports by other researchers and is, overall, in line with the conception of dichotomous role of STINGpathway in tumor development [28]. The data may also indicate STING's participation in as yet unexplored mechanisms promoting tumor development: for example, c-MYC protooncogene which overexpression is a hallmark of cervical cancer has been recently described

**Figure 7.** (A) The change of STING mRNA expression in PBMC isolated from cervical cancer patients compared to healthy women (controls); mean ± SEM values are shown. (B) The change of STING mRNA expression in cervical

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Taking into account that cervical carcinogenesis can be associated with decreased proportion of STING-expressing T lymphocytes, as well as decreased level of STING protein in both T cell subsets (CD4 and CD8), one may assume the involvement of T cell STING in controlling papillomavirus infection and HPV-induced oncopathology. On the other hand, despite the lower level of STING observed in total CD3CD4 population of patients' peripheral blood lymphocytes, in CD25-positive subpopulation its expression was sustained at levels similar to the control: as CD25 is known to be a Treg marker, as well as a T-activation marker, this may be related to the processes of T cell activation/proliferation, interleukin (IL) 2-signaling, and Т cell death. This assumption can be confirmed by recent findings [14, 15] demonstrating antiproliferative or cell-death promoting activity of STING in TCR-stimulated T cells. Previously, we also showed up-regulation of apoptotic processes in circulating lymphocytes from earlystage cervical cancer patients [23], which was correlated with the expansion of CD25-positive cells (including FoxP3-expressing Treg) prompting further investigation of STING in T cells during virus-related carcinogenesis. Thus, the study of naturally occurred cervical neoplastic pathology that develops as a result of chronic viral infection suggests that STING being a key player in modulation of innate immune reactions may have an essential role in Т cell functions. During the development of infection-related cancer, the importance of this specific role can be realized through redistribution of STING levels in different Т cell subsets. Oppositely directed changes in STING expression observed in different compartments—blood T lymphocytes and neoplastic tissue—may illustrate the putative dual role of STING in virus-related

as an essential transcription factor for the STING gene [29]).

neoplastic lesions; group mean values are depicted as horizontal bars; ns—not significant.

**Figure 6.** Percentage of peripheral blood lymphocytes stained positively for STING in patients with precancerous cervical lesions or cancer (stage 0-IA, n = 20) vs. healthy donors (control, n = 15). Mean ± SEM values are shown, ns—not significant.

level showed a slight decrease compared to the control group (p > 0.05, U-test; **Figure 7A**) suggesting the need for T cell (CD4/CD8) separation in further analysis. STING-mRNA expression was also assessed in samples of HPV-negative morphologically normal epithelium (control), HPV-positive precancerous lesions of the cervix, carcinoma in situ and microinvasive carcinoma (relative to four genes—EEF1A1, ACTB, GAPDH, and RPLP0—taken as endogenous controls due to their proved constitutive expression in cervical tissues [27]) (**Figure 7B**). In contrast to lymphocytes, a considerable (up to 50%) proportion of pathological samples

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**Figure 7.** (A) The change of STING mRNA expression in PBMC isolated from cervical cancer patients compared to healthy women (controls); mean ± SEM values are shown. (B) The change of STING mRNA expression in cervical neoplastic lesions; group mean values are depicted as horizontal bars; ns—not significant.

in each patient group showed elevated STING expression as compared with normal noninfected epithelium (though the group mean values did not differ statistically). Up-regulation of STING at early stages of cervical carcinogenesis is consistent with some previous reports by other researchers and is, overall, in line with the conception of dichotomous role of STINGpathway in tumor development [28]. The data may also indicate STING's participation in as yet unexplored mechanisms promoting tumor development: for example, c-MYC protooncogene which overexpression is a hallmark of cervical cancer has been recently described as an essential transcription factor for the STING gene [29]).

Taking into account that cervical carcinogenesis can be associated with decreased proportion of STING-expressing T lymphocytes, as well as decreased level of STING protein in both T cell subsets (CD4 and CD8), one may assume the involvement of T cell STING in controlling papillomavirus infection and HPV-induced oncopathology. On the other hand, despite the lower level of STING observed in total CD3CD4 population of patients' peripheral blood lymphocytes, in CD25-positive subpopulation its expression was sustained at levels similar to the control: as CD25 is known to be a Treg marker, as well as a T-activation marker, this may be related to the processes of T cell activation/proliferation, interleukin (IL) 2-signaling, and Т cell death. This assumption can be confirmed by recent findings [14, 15] demonstrating antiproliferative or cell-death promoting activity of STING in TCR-stimulated T cells. Previously, we also showed up-regulation of apoptotic processes in circulating lymphocytes from earlystage cervical cancer patients [23], which was correlated with the expansion of CD25-positive cells (including FoxP3-expressing Treg) prompting further investigation of STING in T cells during virus-related carcinogenesis. Thus, the study of naturally occurred cervical neoplastic pathology that develops as a result of chronic viral infection suggests that STING being a key player in modulation of innate immune reactions may have an essential role in Т cell functions. During the development of infection-related cancer, the importance of this specific role can be realized through redistribution of STING levels in different Т cell subsets. Oppositely directed changes in STING expression observed in different compartments—blood T lymphocytes and neoplastic tissue—may illustrate the putative dual role of STING in virus-related

level showed a slight decrease compared to the control group (p > 0.05, U-test; **Figure 7A**) suggesting the need for T cell (CD4/CD8) separation in further analysis. STING-mRNA expression was also assessed in samples of HPV-negative morphologically normal epithelium (control), HPV-positive precancerous lesions of the cervix, carcinoma in situ and microinvasive carcinoma (relative to four genes—EEF1A1, ACTB, GAPDH, and RPLP0—taken as endogenous controls due to their proved constitutive expression in cervical tissues [27]) (**Figure 7B**). In contrast to lymphocytes, a considerable (up to 50%) proportion of pathological samples

**Figure 6.** Percentage of peripheral blood lymphocytes stained positively for STING in patients with precancerous cervical lesions or cancer (stage 0-IA, n = 20) vs. healthy donors (control, n = 15). Mean ± SEM values are shown, ns—not

**Figure 5.** (A) The change of STING protein levels in major subsets of peripheral blood T cells from patients with precancerous cervical lesions or cancer (stage 0-IA, n = 20) relative to the control group of healthy donors (n = 15). (B) The ratio of the relative STING expression level in different T cell populations. Mean ± SEM values are displayed; p-value

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significant.

was assessed by U-test.

carcinogenesis, which, in turn, may represent an important point in prognosing therapeutic outcome of STING stimulation. While administration of STING agonists may occur beneficial, for example, for patients with T cell-derived cancer (or other lymphoproliferative disorders) due to promotion of apoptosis in malignant T cells [16], mobilization of STING activity in solid tumors may have an opposite effect due to increased apoptosis of T effectors.

differentiate into functionally impaired dendritic cells or M2-polarized macrophages to maintain pro-inflammatory environment. Despite they have mature phenotype, dendritic cells are not able to migrate to the lymph nodes to initiate adaptive response due to the lack of appropriate homing receptors; instead they accumulate within cervical cancer stroma and secrete protumorigenic and Th2-polarizing factors. Cervical cancer-infiltrating M2-macrophages not only fail to produce IFNs at levels required for T cell activation and proliferation, but also express ligands for the immune checkpoint molecules, for example PD-1L, thereby promoting cytotoxic T cell exhaustion [6, 32–34]. Interestingly, according to Swangphon et al., cervical cancer patients exhibit altered ratio of M1/M2-polarized (CD64+/CD163+) monocytes not only at the local level, but in systemic circulation as well; notably, circulating M1/M2 ratio was shown to be correlated with the number of stroma- or peritumoral area-infiltrating M2-macrophages (CD163+), and with severity of the disease [35]. Similarly, cervical cancer patients displayed increased numbers of circulating dendritic cells (CD11b+) expressing PD-1L [36]. Moreover, an increase in the number of tumor-promoting M2-macrophages/ monocytes has been found to occur not only locally, i.e., in the tumor site, or systemically, i.e., in circulation, but also in tumor-draining lymph nodes (TDLN) of cervical cancer patients implying that the number of PD-1L+ M2-macrophages and metastasis are interrelated; this association allows to suppose that metastasizing cancer cells have the ability to recruit CD14+ monocytes and drive their conversion into M2-macrophages

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

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further contributing to the expansion of highly suppressive Treg cells [34].

Progression of precursor lesions into cervical cancer is also accompanied by an increase in the number of infiltrating neutrophils (TANs) displaying suppressive phenotype. A negative correlation found between the amount of TANs and CD8 T cells in high-grade lesions (cervical intraepithelial neoplasia grade 3, CIN3) or cervical cancer samples suggests that TANs can potentially contribute to inhibition of T cell activity and thereby facilitate tumor growth [32]. This assumption was confirmed experimentally in *in vitro* cell system using co-cultures of SiHa-spheroids, *ex vivo*-stimulated T lymphocytes, and neutrophils, with the ratio of T cell/neutrophil numbers appeared to be the determining factor for the degree of suppression of T cell proliferation, their expression of activation markers, secretion of IFNγ, and cytotoxic activity against SiHa cells [32]. At the systemic level—in the peripheral blood of cervical cancer patients—higher frequency of immature low density neutrophils has been also revealed, with elevated serum levels of granulocyte colony stimulating factor (G-CSF) discovered not only in cervical cancer patients, but also in women with precursor lesions (CIN2-3). Furthermore, patients diagnosed with cervical cancer are characterized by a systemic increase in the frequency of the tolerogenic monocyte-derived dendritic cells (MoDCs), the differentiation of which is modulated by G-CSF: MoDCs that were differentiated from monocytes taken from patients with CIN3 or cervical cancer and showing higher serum level of G-CSF were able to significantly more intensively inhibit proliferation of T cells from healthy donors and to promote Treg differentiation in the *ex vivo* system [32]. The effect of cervical neoplastic lesions on the process of MoDCs differentiation (expression of maturation markers, the profile of secreted cytokines) has been also demonstrated in a study by Lopes et al. [37]. Altogether, these data once again prove that early neoplastic lesions can be accompanied by systemic deviations in innate immunity, which in turn can influence redistribution of innate and adaptive cell populations and their interactions with each other within the tumor locus. The entirety of systemic and local immune changes is also an important point to consider when developing antitumor therapies based on adoptive DC transfer, because it is obviously these changes that determine the absence of the desired therapeutic effect (such developments

Summarizing, it is worth noting that the abundance of STING in T cells may imply; on one hand, their engagement in the innate immune mechanisms (as was revealed by a study of Larkin and co-authors who observed induction of intact antiviral IFN-I response in mouse T cells upon stimulation with STING agonists [14]) or, on the other hand, the plausibility of noncanonical functions exerted by human STING in cells of the adaptive immune system, these issues to be further investigated in the norm and in various pathological states, including virus-induced cervical cancer. In conclusion, it is worth mentioning that such noncanonical activity of STING, specifically, ability to switch on the apoptotic pathway has been unraveled not only in T cells, but in murine B lymphocytes (normal and malignant) as well [30]. However, in another study, the expression of STING in human B cells could be detected only upon Epstein-Barr virus-mediated transformation, while normal B lymphocytes were unable to elicit IFN-I response upon treatment with STING agonists due to the absence of STING expression [31]. Regarding other types of lymphocytes, for instance, NK cells, there is limited or no information. According to our flow cytometry data, the level of STING protein in circulating natural killer cells from patients with cervical carcinoma in situ is notably lower than in CD3CD4 and CD3CD8 (ΔMFI for CD3negCD16pos population was 2.16±0.16), but nonetheless 35 ± 4% of NK cells appear to be STING-positive suggesting potential involvement of STING in NK cell functions.
