**5. Regulatory T and B cells and immune checkpoint molecules in cervical cancer: at the crossroads of immune suppression mechanisms**

Mobilization of intrinsic immune checkpoint mechanisms by a tumor to restrict antitumor immune response is one of the topical issues currently discussed; the phenomenon of effector cell exhaustion and the expression of checkpoint markers have been described for various innate/ acquired immune cell populations, including innate-like cells. Regarding cervical cancer, one can observe an avalanche of new data emerged in recent 2 years on the expression of immune checkpoint markers, first of all PD-1/PD-1L, a hallmark of T cell exhaustion caused by chronic antigenic stimulation [66], as well as other members of B7 and CD28 protein families (e.g., B7-H3 [67] and B7-H4 [68]). For example, patients with CIN or cervical cancer show increased expression of PD-1 both in infiltrating lymphocytes and macrophages (TAMs) [69, 70], as well as in circulating CD4 and CD8 T cells [36], and, furthermore, in the sentinel lymph nodes [71]. Cervical neoplastic cells are considered as the primary source of PD-1 Ligand (PD-1L) [69, 70], with HPV16-Е7 oncoprotein proved to be the driving force for elevated PD-L1 expression [72] and copy number gains of PD-L1 gene being one of the putative underlying reasons [73, 74]. Tumor-infiltrating and stromal M2 macrophages are another such source [34]. Finally, there is one more important source of PD-L1 among adaptive immune cells represented by regulatory T cells (Treg); a correlation between PD-L1 expression and FoxP3+Treg was reported by Ma et al. [75]. Moreover, CD4CD25 Tregs are able to upregulate PD-1 expression in patients with CIN/ cervical carcinoma [36], which, however, does not result in Treg exhaustion, but, conversely, favors upregulation of their immunosuppressive activity. Lastly, it has been reported that regional lymph nodes from stage IB1 cervical cancer patients, along with elevated PD-1, have increased expression of FoxP3 Treg-marker, which may shed light on the establishment of pre-metastatic niches [71], as well as on systemic expansion of suppressive mechanisms Tregs are engaged in.

Several studies have previously reported on increased frequency of circulating CD4 Tregs at initial stages of cervical cancer development [76–78]. Furthermore, we have recently confirmed systemic expansion of Tregs within not only CD4 cell subset, but within CD8 subset as well, at as early as preinvasive and microinvasive cancer (**Figure 10**); we have also revealed correlations between the number of circulating Tregs and the T cell expression of markers of apoptosis, whose induction is supposed to be one of the mechanisms mediating exhaustion of T effector pool during cervical cancer progression [23]. In parallel, the search of new mechanisms providing conditions for Treg expansion during the course of cervical cancer progression is continued: for example, it has been recently found that cervical cancer cells, as well as mesenchymal stromal cells isolated from cervical tumor tissue can upregulate CD73 ectonucleotidase to generate high amounts of adenosine, a potent inducer of Treg differentiation and recruitment [79, 80].

Apart from regulatory T cells, the potential involvement of regulatory B lymphocytes (Breg) in cervical cancer promotion should not be ignored. This can be supported by the results obtained

**5. Regulatory T and B cells and immune checkpoint molecules in cervical cancer: at the crossroads of immune suppression** 

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

Mobilization of intrinsic immune checkpoint mechanisms by a tumor to restrict antitumor immune response is one of the topical issues currently discussed; the phenomenon of effector cell exhaustion and the expression of checkpoint markers have been described for various innate/ acquired immune cell populations, including innate-like cells. Regarding cervical cancer, one can observe an avalanche of new data emerged in recent 2 years on the expression of immune checkpoint markers, first of all PD-1/PD-1L, a hallmark of T cell exhaustion caused by chronic antigenic stimulation [66], as well as other members of B7 and CD28 protein families (e.g., B7-H3 [67] and B7-H4 [68]). For example, patients with CIN or cervical cancer show increased expression of PD-1 both in infiltrating lymphocytes and macrophages (TAMs) [69, 70], as well as in circulating CD4 and CD8 T cells [36], and, furthermore, in the sentinel lymph nodes [71]. Cervical neoplastic cells are considered as the primary source of PD-1 Ligand (PD-1L) [69, 70], with HPV16-Е7 oncoprotein proved to be the driving force for elevated PD-L1 expression [72] and copy number gains of PD-L1 gene being one of the putative underlying reasons [73, 74]. Tumor-infiltrating and stromal M2 macrophages are another such source [34]. Finally, there is one more important source of PD-L1 among adaptive immune cells represented by regulatory T cells (Treg); a

**Figure 9.** Percentage of peripheral blood T cells with NK-like phenotype in patients with CIN3 or microinvasive carcinoma (St IA) and healthy controls. Lymphocyte populations of interest were defined according to CD3/CD56

**mechanisms**

expression levels (gates P1-P3).

**Figure 10.** The frequencies of peripheral blood Treg lymphocytes in patients with CIN3 or microinvasive carcinoma (St IA) and healthy controls. (A) CD4 Tregs were gated according to the level of CD25, CD127, and FoxP3 expression; gating of CD8 Tregs was performed in a similar way. (B) The change in the frequency of circulating CD4 regulatory cells in patients compared to healthy donors. (C) The change in the frequency of circulating CD8 regulatory cells in patients compared to healthy donors: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 (U-test).

by Tang et al. who used mouse model of HPV-related cancer to demonstrate that Bregs accumulate in tumor-draining lymph nodes, have altered phenotype (specifically, altered expression of cell surface markers, such as MHC II, PD-L1, and CD39), exhibit high regulatory potency, thus fostering tumor growth [81]. In humans, many types of solid tumors were found to be accompanied with increased numbers of both tumor-infiltrating and circulating Bregs capable of producing suppressor cytokines (e.g., IL-10) and immune checkpoint ligands, thus impairing T cell function (see reviews [82, 83]), suggesting this issue to be investigated for cervical cancer patients.

CD Cluster of differentiation

CIN Cervical intraepithelial neoplasia

DAMP Damage-associated molecular patterns

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

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

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G-CSF Granulocyte-colony stimulating factor

cGAMP Cyclic GMP-AMP

cGAS cGAMP synthase

DC Dendritic cells

IFN-I Type I interferon

IL Interleukin

HPV Human papillomavirus

IRF Interferon regulatory factor

MFI Mean fluorescence intensity

MDSC Myeloid-derived suppressor cells

MHC Major histocompatibility complex

MoDC Monocyte-derived dendritic cells

PBL Peripheral blood lymphocytes

PBMC Peripheral blood monocyte cells

PRR Pattern recognition receptor

SEM Standard error of the mean

STING Stimulator of Interferon Genes

TAM Tumor-associated macrophages

TAN Tumor-associated neutrophils

TDLN Tumor-draining lymph nodes

TGFβ Transforming growth factor beta

TCR T cell receptor

MAb Monoclonal antibody

NK Natural killer cells

NKT Natural killer-like cells

## **6. Conclusion**

Further ways to develop approaches for the treatment of HPV-associated malignancies, including cervical cancer, belong to the area of combined therapies, where particular attention is to be paid to restoration the effectiveness of innate mechanisms of immune response, including those trigged by PRRs (e.g., STING). PRR agonists are expected to serve potent adjuvant function; another promising area is the use of agonists to stimulate NK, NKT, and γδT receptors. Despite substantial progress, there is clear understanding that stimulation of innate immune cells "per se" is senseless without concomitant inhibition of immunosuppressive factors (such as inhibitory molecules of immune checkpoint or other Treg-associated factors). Therefore, as illustrated by recent findings summarized in the chapter, there is obvious need for continuing comprehensive characterization of functional diversity of innate immune cells that organize cervical cancer immune regulatory network, exploration of noncanonical functions of innate immunity mediators, identification of precise resources of immune suppression and assessments of local and systemic changes in immune parameters.
