**4. Innate-like T lymphocytes: pivotal players in the tumor: immunity interplay**

#### **4.1. The emerging role of NK-like T cells (NKT) and γδT lymphocytes in cervical cancer progression**

Since recently, a heterogeneous group of innate-like T lymphocytes linking the two branches of immunity, innate and adaptive, is being increasingly acknowledged as a valuable source of novel opportunities for antitumor therapies development. This was facilitated by increasing realization of the pivotal role of innate-like T cells in tumor immune surveillance and their unique ability to recognize cancerous and virus-infected cells in a highly specific, though MHC-unrestricted, manner. Similar to innate immune cells, they are equipped with a rich set of germline-encoded receptors conferring them ability to undergo rapid activation upon interaction with ectopically expressed or stress-associated molecules on the surface of target cells. In addition, like conventional αβT cells, innate-like T lymphocytes (NKT and γδT) express T cell receptors (TCR), although their repertoire differs from αβT cells. The range of antigens the innate-like lymphocytes' TCRs are able to recognize is defined by their structural properties (for example, lipid antigens in the case of NKT cells or phosphoantigens in the case of γδT cells) and therefore is thought to be restricted and universal; at the same time, these antigens are present within a plenty of natural ligands, which is doubtless advantageous from a therapeutic standpoint. Quick activation in response to antigenic exposure followed by intense production of a broad range of cytokines is another valuable characteristic of innate-like lymphocytes they have in common with typical innate lymphocytes; it is known, for instance, that even in the absence of stimulation NKT cells permanently stay in pre-activated state. That is why innate-like lymphocytes supposedly perform "guarding" functions by being the first to respond efficiently to pathological changes (infection, transformation) and stimulate further activation of dendritic cells and adaptive response. However, in spite of their apparent beneficial properties, there are several features that greatly impede potential manipulations of innate-like lymphocytes, among them are: high structural and functional population heterogeneity, low abundance, heterogeneous distribution of different subpopulations in tissue and blood compartments, ability to provoke chronic inflammation and to secrete not only Th1-cytokines, but Th2 as well. If structural heterogeneity of innate-like lymphocytes is defined by their receptor repertoire, their functional heterogeneity is believed to be driven by polarizing factors coming from the environment. Importantly, conclusions about existing functional subtypes of innate-like lymphocytes were made based mostly on the results of in vitro stimulation [53–55].

Like conventional Т lymphocytes, NKT cells express αβTCR, but can undergo activation only on interaction with lipid antigens presented by CD1b (a nonpolymorphic MHC-I-like molecule). In spite of such a relatively narrow specificity, NKT cells however exhibit an important feature ability for TCR-independent activation upon stimulation with proinflammatory cytokines IL-12, IL-18, IL-25, and IL-23. According to the structure and binding specificity of TCRs, two NKT subsets can be distinguished: NKT-I, or iNKT—invariant NKT cells (with α-galactosylceramide being a prototypic ligand), and NKT-II cells—variant NKT having less restricted specificity. NKT-II cells are thought to be the most prevalent NKT subset in humans (in contrast to, for example, mice, where NKT-I cells are known to be more abundant), although their identification and characterization is still a challenging task due to the lack of distinctive NKT-II markers or agonists specifically targeting their receptors. In general, following the results of *in vivo* modeling of various cancers, NKT-I cells have been associated with the protective antitumor response, while NKT-II have been implicated in immunosuppression/immunoregulation and tumor promotion. The mechanisms of antitumor activity of NKT-I cells consist in their ability for both direct tumor lysis and generation of copious amounts of IFNγ (along with other Th1 cytokines) required for recruitment and activation/full maturation of APC, CD8 cytotoxic T lymphocytes, and NK cells. Immunosuppressive effect of NKT-II cells is thought to be due to their ability to produce high levels of IL-4 and IL-13 that shift immune response towards Th2 type. Nevertheless, this functional dichotomy is at present actively debated, and there is growing conviction that it is not so firmly associated with NKT-I or -II subset; rather, it is determined by the context (for example, tissue location) or microenvironment where activation of NKT cells occurs [53]. (Due to limited space, in our characteristic of NKT cells and γδT cells, here and below we refer to several recently published comprehensive reviews that contain links to original papers).

**4. Innate-like T lymphocytes: pivotal players in the tumor: immunity** 

control group are marked with asterisk: \*p < 0.05, \*\*p < 0.01 (U-test).

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

**Figure 8.** Percentage of peripheral blood CD56bright NK cells and CD56dim/CD56bright ratio within circulating NK cell population in patients (n = 30 for CIN3/stage 0, n = 15 for stage IA) vs. healthy controls (n = 30) as measured by flow cytometry. Lymphocytes were gated for CD3-negativity (a diagram on the left) and a population of interest was defined according to CD16/CD56 membrane expression levels (gates P1–P4). Here and below, individual values are shown as dots; bars correspond to the mean ± SEM values; statistically significant difference between the patient group and the

**4.1. The emerging role of NK-like T cells (NKT) and γδT lymphocytes in cervical** 

Since recently, a heterogeneous group of innate-like T lymphocytes linking the two branches of immunity, innate and adaptive, is being increasingly acknowledged as a valuable source of novel opportunities for antitumor therapies development. This was facilitated by increasing realization of the pivotal role of innate-like T cells in tumor immune surveillance and their unique ability to recognize cancerous and virus-infected cells in a highly specific, though MHC-unrestricted, manner. Similar to innate immune cells, they are equipped with a rich set of germline-encoded receptors conferring them ability to undergo rapid activation upon interaction with ectopically expressed or stress-associated molecules on the surface of target cells. In addition, like conventional αβT cells, innate-like T lymphocytes (NKT and γδT) express T cell receptors (TCR), although their repertoire differs from αβT cells. The range of antigens the innate-like lymphocytes' TCRs are able to recognize is defined by their structural properties (for example, lipid antigens in the case of NKT cells

**interplay**

**cancer progression**

In spite of the relatively low abundance of NKT cells, there is constantly growing body of evidence showing this cell population undergoes quantitative and phenotypic changes (both in peripheral blood and within the tumor locus) in patients with different types of cancer, however there is only scares information available for cervical cancer. It has been found that HPV can escape from NKT cell-mediated CD1d-restricted recognition of infected keratinocytes and low-grade cervical neoplastic lesions via HPV-E5 dependent inhibition of CD1d expression (while normal keratinocytes express high levels of CD1d molecule) [56]. Despite this evasion mechanism, CIN2-3 lesions were shown to be associated with increased numbers of infiltrating iNKT, with these numbers being higher for HPV-positive lesions than for HPVnegative [57]. Elevated frequency of circulating NKT have been revealed in peripheral blood of women with CIN1 and HPV infection, compared to the control group or HPV-positive women without signs of neoplastic abnormalities [58]. It can be inferred from these findings that the population of NKT cells may undergo early changes upon persistent HPV infection and progressing neoplasia, although there is no data available for more advanced stages of the disease.

for the development of adoptive cell transfer therapy combined with immune checkpoints blockade or neutralization of other immune-suppressive factors [55, 61]. At the same time, the results of preclinical studies and attempts to translate them to clinical settings explicitly point to our insufficient knowledge of the role of innate-like lymphocytes and the mechanisms,

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CD56 is a natural killer prototypic marker, but, apart from NK cells, its expression is shared by T lymphocytes (NKТ and γδT) and is commonly considered as a marker of an activated state, NK-like cytotoxicity and IFNγproduction [62]. Accordingly, CD3+CD56+ population comprises of both NKТ and γδT lymphocytes, but within this population, a CD3bright subset can be observed [63]. Studies on phenotyping of CD3bright subpopulation have identified it as γδT lymphocytes [63, 64]. Furthermore, Paget and co-authors [64] established mouse CD3bright γδT cells were identical to Vδ1 sub-lineage (Vγ6/Vδ1+ TCR) and possessed high IL-17-producing capacity; the high CD3 expression (CD3bright phenotype) could hence be considered as a surrogate marker of γδT identity. In humans, the population of circulating CD3brightCD56+ cells has been analyzed in patients with chronic hepatitis B infection: it has been shown that, despite increased numbers, their activity and phenotype are substantially impaired, this impairment includes down-modulation of IFNγ and LAMP1 expression (i.e., markers of antiviral and killing activity) and, conversely, up-regulation of NKG2A [65].

Given, the described data on the ability of CD3bright T cells to respond to inflammation and chronic infection observed either in model animals or in humans, in the norm or under pathological conditions, we decided to examine whether changes of the frequency of this cell population could be detected in the circulation of patients at early stages of cervical cancer progression (**Figure 9**). Using clone UCHT1 of anti-human CD3 MAb, we were able to clearly identify subpopulation of CD3bright lymphocytes in peripheral blood of CUN3/cervical cancer patients and the controls. In spite of the relatively wide range of individual values in all studied groups, a trend towards a decreased number of circulating CD3brightCD56+ cells (p > 0.05) was observed for women with microinvasive carcinoma (gate P1, **Figure 9**); at the same time, no difference in the frequency of cells falling within gate P2 and the total frequency of CD3+CD56+ (including CD3+CD16+/-CD56+) cells was revealed (data not shown). Then, within the CD3bright population, we also analyzed the expression of CD16, a marker of antibody-mediated cytotoxicity, but did not find any significant difference between the controls and the patients groups (data not shown). According to Lambert et al., CD3bright T cells (i.e., γδT) do not express CD4, but express low levels of CD8 [63]. We compared the frequencies of CD3brightCD8low cells (gate P3) between the study groups, but again did not observe any difference. Therefore, in contrast to regulatory NK cells, the combination of CD3/ CD16/CD56 markers is not sufficient to show if there are any significant changes occurring within the population of circulating innate-like T cells in early-stage cervical cancer. Although these results cannot be compared with the results reported by Pita-Lopez et al., who used the same combination of CD markers to analyze blood samples from women with low-grade lesions (CIN1), nevertheless, observations made for CD3brightCD56 cells, along with published data mentioned above underline the need for continuing investigation into innate-like lymphocytes at various stages of cervical cancer development and progression with the use of lineage-specific (e.g., anti-TCR) antibodies.

**4.2. Analysis of CD3+CD56+ population and its CD3bright subset in PBMC from** 

whereby they contribute to cancer progression [53, 55].

**early-stage cervical cancer patients**

γδТ lymphocytes differ from both conventional T cells and NKT cells in their TCR chains composition and ability to recognize phosphoantigens, while many other features characteristic of NKT cells are shared by γδT as well, specifically: rapid activation, direct cytotoxicity against infected or transformed cells, reliance on natural killer receptors that enable fast (MHC-independent) response to stress-related ligands expressed on the surface of cancer cells, strong regulatory properties and ability to modulate activity of other immune cells via production of a wide range of cytokines. Further, similar to NKT cells, γδТ also demonstrate functional heterogeneity (polarization) with regard to antitumor response, with this heterogeneity partially overlapping with the structural features of γδTCR, but nevertheless being mostly driven by differential environmental stimulation, as was mentioned for NKT cells. In humans, Vδ2 T cells were found to be the most frequent subpopulation of peripheral blood γδТ cells (70%); Vδ1 T cells constitute the remaining 30% of γδТ in circulation, although they represent the dominant γδТ subset in epithelial and some other tissues. Antitumor activity of Vδ2 T cells is attributed to not only their ability to directly recognize (via congenital receptors) and kill tumor cells, but also their ability to effectively cross-present antigens to CD8 αβТ effectors and NKT cells, as well as facilitate DC maturation and co-stimulate cytolytic activity of NK cells. Pro-tumor role of Vδ1 T cells can be explained by their IL-17-producing ability; at the same time, however, these cells show unique specificity for B7-H6 molecule expressed exclusively on tumor cells, being able thereby to exert antitumor effect. Immunoregulatory (suppressive) function of γδT cells in antitumor immunity is thought to be mediated by IL-10 and TGF-β, or adenosine secreted by tumor-infiltrating γδT cells [54, 55].

The impact of γδT cells on pathogenesis of cervical cancer is largely unexplored. Gosmann and co-authors analyzed total population of CD45+IL-17+ cells infiltrating CIN2-3 lesions and found them to be represented by not only CD3CD4 T helpers (Th17), but also by γδT cells, although the percentage of γδT cells was significantly lower than that of Th17 [59]; this observation may indicate their putative involvement in the promotion of proinflammatory suppressive microenvironment as CIN progresses to invasive cancer. The cytotoxic activity of γδT cells isolated from PBMC against cervical cancer cell lines (HeLa, SiHa, and CaSki) pre-treated with bisphosphonate pamidronate was also confirmed in [60]. A vast amount of clinical data on the role of γδT cells in viral infections, as well as their correlation with cancer prognosis allows speculation on γδT cell involvement in pathogenesis of virus-associated cervical cancer. Whether this cell population experiences any changes at different stages of cervical cancer, and if so, in which tissue compartments or depending on which clinical-pathological parameters—remains an open question.

Taken together, innate and innate-like (NK, NKT, and γδT) lymphocytes proved to have nonredundant functions in antitumor immune response, which makes them attractive objects for the development of adoptive cell transfer therapy combined with immune checkpoints blockade or neutralization of other immune-suppressive factors [55, 61]. At the same time, the results of preclinical studies and attempts to translate them to clinical settings explicitly point to our insufficient knowledge of the role of innate-like lymphocytes and the mechanisms, whereby they contribute to cancer progression [53, 55].

#### **4.2. Analysis of CD3+CD56+ population and its CD3bright subset in PBMC from early-stage cervical cancer patients**

HPV can escape from NKT cell-mediated CD1d-restricted recognition of infected keratinocytes and low-grade cervical neoplastic lesions via HPV-E5 dependent inhibition of CD1d expression (while normal keratinocytes express high levels of CD1d molecule) [56]. Despite this evasion mechanism, CIN2-3 lesions were shown to be associated with increased numbers of infiltrating iNKT, with these numbers being higher for HPV-positive lesions than for HPVnegative [57]. Elevated frequency of circulating NKT have been revealed in peripheral blood of women with CIN1 and HPV infection, compared to the control group or HPV-positive women without signs of neoplastic abnormalities [58]. It can be inferred from these findings that the population of NKT cells may undergo early changes upon persistent HPV infection and progressing neoplasia, although there is no data available for more advanced stages of the disease. γδТ lymphocytes differ from both conventional T cells and NKT cells in their TCR chains composition and ability to recognize phosphoantigens, while many other features characteristic of NKT cells are shared by γδT as well, specifically: rapid activation, direct cytotoxicity against infected or transformed cells, reliance on natural killer receptors that enable fast (MHC-independent) response to stress-related ligands expressed on the surface of cancer cells, strong regulatory properties and ability to modulate activity of other immune cells via production of a wide range of cytokines. Further, similar to NKT cells, γδТ also demonstrate functional heterogeneity (polarization) with regard to antitumor response, with this heterogeneity partially overlapping with the structural features of γδTCR, but nevertheless being mostly driven by differential environmental stimulation, as was mentioned for NKT cells. In humans, Vδ2 T cells were found to be the most frequent subpopulation of peripheral blood γδТ cells (70%); Vδ1 T cells constitute the remaining 30% of γδТ in circulation, although they represent the dominant γδТ subset in epithelial and some other tissues. Antitumor activity of Vδ2 T cells is attributed to not only their ability to directly recognize (via congenital receptors) and kill tumor cells, but also their ability to effectively cross-present antigens to CD8 αβТ effectors and NKT cells, as well as facilitate DC maturation and co-stimulate cytolytic activity of NK cells. Pro-tumor role of Vδ1 T cells can be explained by their IL-17-producing ability; at the same time, however, these cells show unique specificity for B7-H6 molecule expressed exclusively on tumor cells, being able thereby to exert antitumor effect. Immunoregulatory (suppressive) function of γδT cells in antitumor immunity is thought to be mediated by IL-10 and TGF-β, or adenosine secreted by tumor-infiltrating γδT cells [54, 55]. The impact of γδT cells on pathogenesis of cervical cancer is largely unexplored. Gosmann and co-authors analyzed total population of CD45+IL-17+ cells infiltrating CIN2-3 lesions and found them to be represented by not only CD3CD4 T helpers (Th17), but also by γδT cells, although the percentage of γδT cells was significantly lower than that of Th17 [59]; this observation may indicate their putative involvement in the promotion of proinflammatory suppressive microenvironment as CIN progresses to invasive cancer. The cytotoxic activity of γδT cells isolated from PBMC against cervical cancer cell lines (HeLa, SiHa, and CaSki) pre-treated with bisphosphonate pamidronate was also confirmed in [60]. A vast amount of clinical data on the role of γδT cells in viral infections, as well as their correlation with cancer prognosis allows speculation on γδT cell involvement in pathogenesis of virus-associated cervical cancer. Whether this cell population experiences any changes at different stages of cervical cancer, and if so, in which tissue compartments or depending on which clinical-pathological parameters—remains an open question.

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

Taken together, innate and innate-like (NK, NKT, and γδT) lymphocytes proved to have nonredundant functions in antitumor immune response, which makes them attractive objects CD56 is a natural killer prototypic marker, but, apart from NK cells, its expression is shared by T lymphocytes (NKТ and γδT) and is commonly considered as a marker of an activated state, NK-like cytotoxicity and IFNγproduction [62]. Accordingly, CD3+CD56+ population comprises of both NKТ and γδT lymphocytes, but within this population, a CD3bright subset can be observed [63]. Studies on phenotyping of CD3bright subpopulation have identified it as γδT lymphocytes [63, 64]. Furthermore, Paget and co-authors [64] established mouse CD3bright γδT cells were identical to Vδ1 sub-lineage (Vγ6/Vδ1+ TCR) and possessed high IL-17-producing capacity; the high CD3 expression (CD3bright phenotype) could hence be considered as a surrogate marker of γδT identity. In humans, the population of circulating CD3brightCD56+ cells has been analyzed in patients with chronic hepatitis B infection: it has been shown that, despite increased numbers, their activity and phenotype are substantially impaired, this impairment includes down-modulation of IFNγ and LAMP1 expression (i.e., markers of antiviral and killing activity) and, conversely, up-regulation of NKG2A [65].

Given, the described data on the ability of CD3bright T cells to respond to inflammation and chronic infection observed either in model animals or in humans, in the norm or under pathological conditions, we decided to examine whether changes of the frequency of this cell population could be detected in the circulation of patients at early stages of cervical cancer progression (**Figure 9**). Using clone UCHT1 of anti-human CD3 MAb, we were able to clearly identify subpopulation of CD3bright lymphocytes in peripheral blood of CUN3/cervical cancer patients and the controls. In spite of the relatively wide range of individual values in all studied groups, a trend towards a decreased number of circulating CD3brightCD56+ cells (p > 0.05) was observed for women with microinvasive carcinoma (gate P1, **Figure 9**); at the same time, no difference in the frequency of cells falling within gate P2 and the total frequency of CD3+CD56+ (including CD3+CD16+/-CD56+) cells was revealed (data not shown). Then, within the CD3bright population, we also analyzed the expression of CD16, a marker of antibody-mediated cytotoxicity, but did not find any significant difference between the controls and the patients groups (data not shown). According to Lambert et al., CD3bright T cells (i.e., γδT) do not express CD4, but express low levels of CD8 [63]. We compared the frequencies of CD3brightCD8low cells (gate P3) between the study groups, but again did not observe any difference. Therefore, in contrast to regulatory NK cells, the combination of CD3/ CD16/CD56 markers is not sufficient to show if there are any significant changes occurring within the population of circulating innate-like T cells in early-stage cervical cancer. Although these results cannot be compared with the results reported by Pita-Lopez et al., who used the same combination of CD markers to analyze blood samples from women with low-grade lesions (CIN1), nevertheless, observations made for CD3brightCD56 cells, along with published data mentioned above underline the need for continuing investigation into innate-like lymphocytes at various stages of cervical cancer development and progression with the use of lineage-specific (e.g., anti-TCR) antibodies.

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

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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

**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).

on systemic expansion of suppressive mechanisms Tregs are engaged in.

**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 expression levels (gates P1-P3).
