**3. Cellular component of innate immunity (natural killer lymphocytes, myeloid cell populations): its role in regulation of T cell-mediated antitumor/antiviral immunity**

#### **3.1. Regulatory functions of innate immune cells in relation to cervical cancer development: current knowledge**

The regulatory role of myeloid cells (monocytes—dendritic cells and macrophages, and especially granulocytes) has been undervalued for a long time; however, recently emerged data have prompted reconsideration of significance of these cells, classically regarded as professional phagocytes or professional APC, in mediating regulatory/suppressor effects of tumor cells on T-effectors [32]. In addition, the systemic effect of local neoplastic lesions on deviations within these innate immune cell populations, which can become detectable even earlier than the distribution of tumor-infiltrating cell populations is changed, is becoming increasingly apparent [32]. In respect of these abnormalities, a number of fundamentally important data have been obtained for cervical cancer.

According to the model described by Smola et al., IL-6 secreted by HPV-transformed cells acts as a triggering factor that leads to multiple impairments in the key functions of myelomonocytic cells during the intraepithelial stage of cervical cancer development. Under the influence of IL-6 and chemokines, myelocytes are actively recruited into the site of neoplasia, where they can 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 further contributing to the expansion of highly suppressive Treg cells [34].

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

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 sug-

solid tumors may have an opposite effect due to increased apoptosis of T effectors.

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

gesting potential involvement of STING in NK cell functions.

**cell-mediated antitumor/antiviral immunity**

**development: current knowledge**

data have been obtained for cervical cancer.

**3. Cellular component of innate immunity (natural killer** 

**lymphocytes, myeloid cell populations): its role in regulation of T** 

**3.1. Regulatory functions of innate immune cells in relation to cervical cancer** 

The regulatory role of myeloid cells (monocytes—dendritic cells and macrophages, and especially granulocytes) has been undervalued for a long time; however, recently emerged data have prompted reconsideration of significance of these cells, classically regarded as professional phagocytes or professional APC, in mediating regulatory/suppressor effects of tumor cells on T-effectors [32]. In addition, the systemic effect of local neoplastic lesions on deviations within these innate immune cell populations, which can become detectable even earlier than the distribution of tumor-infiltrating cell populations is changed, is becoming increasingly apparent [32]. In respect of these abnormalities, a number of fundamentally important

According to the model described by Smola et al., IL-6 secreted by HPV-transformed cells acts as a triggering factor that leads to multiple impairments in the key functions of myelomonocytic cells during the intraepithelial stage of cervical cancer development. Under the influence of IL-6 and chemokines, myelocytes are actively recruited into the site of neoplasia, where they can 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 aimed at overcoming the suppressive impact on DC are conducted using preclinical murine models of cervical cancer, see, for example, [38, 39]). In addition, a study performed by van Meir et al. showed that myeloid cells from cervical cancer patients can systematically respond to radiotherapy (RT): during the course of RT and 3–9 weeks after its completion (regardless the administration of cisplatin), increased frequencies of circulating CD3(-)CD19(-)HLA-DR(+) monocytes as well as CD3(-)CD19(-)HLA-DR(-) MDSCs were detected in parallel with the loss of T cell reactivity and stimulatory capacity of APC in *ex vivo* testing [40].

killing of tumor cells is less clear; on the other hand, CD56bright NK cells are known for their high cytokine and chemokine production capacity (including IFNγ, TNFα, GM-CSF, IL-10, IL-13, CCL3, and CCL4), and immunomodulation of activity of other innate or adaptive immune

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Presently, increasing attention is being paid to CD56bright NK cells as new facts are emerging suggesting that there is no strict functional dichotomy between the so called regulatory CD56bright and cytolytic CD56dim subsets, and that CD56bright cells are capable of acquiring cytotoxicity upon appropriate stimulation with specific combinations of cytokines [45]. Indeed, it has been recently shown that priming of CD56bright NK cells with IL-15 is accompanied by a burst of cytotoxic activity against tumor cells; however, this has only been confirmed so far for hematological malignancies [46]. Upon treatment with different stimuli, CD56bright NK cells exhibit ability of suppressing proliferation of autologous CD4 T cells via both cytotoxic and immunoregulatory mechanisms, e.g., by secreting the immunosuppressive molecule adenosine (these mechanisms are reviewed in detail in [47]). For some types of solid cancers, in particular lung cancer and breast cancer, the proportion of CD56bright cells in a total amount of tumor-infiltrating NK cells was found to be significantly higher than in the corresponding normal tissues, however, they

express low perforin and rather play an immunoregulatory role, but not cytotoxic [48].

**3.2. Analysis of NK cell subpopulations in peripheral blood lymphocytes of early-**

investigation into the regulatory role of NK cells in cervical cancer progression.

Taking into consideration, the proposed model that describes the ability of CD56bright NK cells to circulate among tissues, lymphoid organs, and peripheral blood [45, 48], it can be assumed that altered frequencies of these cells in the blood of cancer patients are highly relevant to immune regulation at the tumor locus. Quantitative assessment of circulating CD56bright NK cell population has been performed for head and neck cancer [49], prostate [50], and breast cancer [51, 52]); we also recently reported our findings concerning circulating NK subsets in women with CIN3 (including carcinoma in situ) and microinvasive carcinoma (stage IA1) of the cervix [23]. Based on the intensity of CD16/CD56 staining, we could distinguish four main subsets of circulating NK cells within CD3-negative lymphocytes (gates P1–P4, **Figure 8**). As expected, we found no significant difference in the frequency of cells within CD16brightCD56dim gate (which encompasses the major pool of circulating cytotoxic NKs), as well as within CD16dim/negCD-56dim and CD16brightCD56neg gates (comprising less abundant populations with poorly established functions) between patients and controls. As opposed to these subsets, a decrease in the frequency of CD16dim/negCD56bright NK cells and, accordingly, higher CD56dim/ CD56bright ratio were observed in cervical cancer patients relative to the control group. We hypothesized this specific alteration reflects a systemic shift in the balance between effector and regulatory NK subsets that occur early in invasive cervical cancer development. One can also speculate this change, along with those described above for M2/M1, neutrophils and MoDC subsets, is part of a complex cervical cancer-related immunoregulatory network. As it is well known that activation of NK cells occurs locally, in our attempt to interpret the obtained data we therefore use the idea that circulating CD56bright NKregs are recruited to the lymphoid tissue (regional lymph nodes) and the primary tumor site, where they are thought to serve as precursors for cytotoxic/effector CD56dim NK cells [45]. This assumption encourages further

**stage cervical cancer patients**

cells is therefore believed to be a key feature of CD56bright NK cell subset.

Unlike neutrophils and suppressor populations of myeloid cells, whose contribution to the progression of solid tumors has only recently come under intense investigation, the functions of natural killer cells have always been considered in the context of cancer immunosurveillance. However, in spite of the fact that for this group of innate lymphoid cells, a detailed spectrum of receptors allowing for recognition of transformed cells has been described and a vast diversity of mechanisms for their cytotoxic action has been established, attempts to use them in anticancer therapy occurred to be unsuccessful—the reasons for this situation are reviewed in [41], and among these reasons are the underappreciated regulatory properties of NK cells implementing via production of a wide range of cytokines, the specificity of which is largely determined by the surrounding molecular context. Nevertheless, recently there has been considerable revival of interest in NK cells brought about by the invention of chimeric antigen receptors (CAR) technology that made possible creation of engineered CAR-NKs with "improved" properties (e.g., increased migrating and proliferating ability, up-regulated expression of activating receptors) for their subsequent adoptive transfer into a cancer patient. Another promising concept seems to be the use of Cord-Blood NK cells that can retain a highly activated phenotype and whose expansion capacity substantially exceeds that of peripheral blood NK cells (successful implementation of this approach in the preclinical model system using cervical cancer cell lines has been recently reported in [42]).

Cervical cancer cells' ability to withstand NK cell-mediated response is clearly confirmed by the observation that the prevalence of NK cells in CD45(+)-infiltrating leukocytes is greatly reduced with the progression of intraepithelial neoplasia to invasive cancer [32]. In addition to the known mechanisms recruited by cervical cancer cells to escape from NK-mediated recognition (including down-regulation of activating NK-cell receptor ligands MICA/B, ULBPs, or aberrant expression of non-classical HLA-G [43]), inhibition of NK cell activity can be driven by intra-tumoral Tregs, as was confirmed in ex vivo experiments with Tregs and NK cells isolated from primary tumors of cervical cancer patients [44]. Whether these negative processes have any influence on circulating NK cells during the development of cervical cancer remains a poorly studied question.

Despite the high phenotypic heterogeneity of NK cells, they can be divided into two subsets depending on the level of expression of CD56 marker: CD56bright and CD56dim. These two populations differ not only phenotypically and functionally—they are differently represented in the systemic circulation and tissues [45]. CD56dim population comprises the vast majority (80– 95%) of peripheral blood NK cells and is characterized by high expression of markers of mature phenotype (including CD16/FcγRIIIa required for activation of antibody-dependent cytotoxicity, perforin and granzyme B cytotoxic proteins); traditionally, this population is associated with anti-tumor response. Unlike CD56dim, CD56bright NK cells represent the minor population in peripheral blood, while in the secondary lymphoid organs and other tissues CD56bright cells account for the majority of peripheral NKs. In addition, they are characterized by the absence or low expression of CD16 (CD16dim/neg) and low cytotoxic activity, so their role in direct killing of tumor cells is less clear; on the other hand, CD56bright NK cells are known for their high cytokine and chemokine production capacity (including IFNγ, TNFα, GM-CSF, IL-10, IL-13, CCL3, and CCL4), and immunomodulation of activity of other innate or adaptive immune cells is therefore believed to be a key feature of CD56bright NK cell subset.

aimed at overcoming the suppressive impact on DC are conducted using preclinical murine models of cervical cancer, see, for example, [38, 39]). In addition, a study performed by van Meir et al. showed that myeloid cells from cervical cancer patients can systematically respond to radiotherapy (RT): during the course of RT and 3–9 weeks after its completion (regardless the administration of cisplatin), increased frequencies of circulating CD3(-)CD19(-)HLA-DR(+) monocytes as well as CD3(-)CD19(-)HLA-DR(-) MDSCs were detected in parallel with the loss of T cell

Unlike neutrophils and suppressor populations of myeloid cells, whose contribution to the progression of solid tumors has only recently come under intense investigation, the functions of natural killer cells have always been considered in the context of cancer immunosurveillance. However, in spite of the fact that for this group of innate lymphoid cells, a detailed spectrum of receptors allowing for recognition of transformed cells has been described and a vast diversity of mechanisms for their cytotoxic action has been established, attempts to use them in anticancer therapy occurred to be unsuccessful—the reasons for this situation are reviewed in [41], and among these reasons are the underappreciated regulatory properties of NK cells implementing via production of a wide range of cytokines, the specificity of which is largely determined by the surrounding molecular context. Nevertheless, recently there has been considerable revival of interest in NK cells brought about by the invention of chimeric antigen receptors (CAR) technology that made possible creation of engineered CAR-NKs with "improved" properties (e.g., increased migrating and proliferating ability, up-regulated expression of activating receptors) for their subsequent adoptive transfer into a cancer patient. Another promising concept seems to be the use of Cord-Blood NK cells that can retain a highly activated phenotype and whose expansion capacity substantially exceeds that of peripheral blood NK cells (successful implementation of this approach in the preclinical model system using cervical cancer cell lines has been recently reported in [42]). Cervical cancer cells' ability to withstand NK cell-mediated response is clearly confirmed by the observation that the prevalence of NK cells in CD45(+)-infiltrating leukocytes is greatly reduced with the progression of intraepithelial neoplasia to invasive cancer [32]. In addition to the known mechanisms recruited by cervical cancer cells to escape from NK-mediated recognition (including down-regulation of activating NK-cell receptor ligands MICA/B, ULBPs, or aberrant expression of non-classical HLA-G [43]), inhibition of NK cell activity can be driven by intra-tumoral Tregs, as was confirmed in ex vivo experiments with Tregs and NK cells isolated from primary tumors of cervical cancer patients [44]. Whether these negative processes have any influence on circulating NK cells during the development of cervical cancer remains a poorly studied question. Despite the high phenotypic heterogeneity of NK cells, they can be divided into two subsets depending on the level of expression of CD56 marker: CD56bright and CD56dim. These two populations differ not only phenotypically and functionally—they are differently represented in the systemic circulation and tissues [45]. CD56dim population comprises the vast majority (80– 95%) of peripheral blood NK cells and is characterized by high expression of markers of mature phenotype (including CD16/FcγRIIIa required for activation of antibody-dependent cytotoxicity, perforin and granzyme B cytotoxic proteins); traditionally, this population is associated with anti-tumor response. Unlike CD56dim, CD56bright NK cells represent the minor population in peripheral blood, while in the secondary lymphoid organs and other tissues CD56bright cells account for the majority of peripheral NKs. In addition, they are characterized by the absence or low expression of CD16 (CD16dim/neg) and low cytotoxic activity, so their role in direct

reactivity and stimulatory capacity of APC in *ex vivo* testing [40].

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

Presently, increasing attention is being paid to CD56bright NK cells as new facts are emerging suggesting that there is no strict functional dichotomy between the so called regulatory CD56bright and cytolytic CD56dim subsets, and that CD56bright cells are capable of acquiring cytotoxicity upon appropriate stimulation with specific combinations of cytokines [45]. Indeed, it has been recently shown that priming of CD56bright NK cells with IL-15 is accompanied by a burst of cytotoxic activity against tumor cells; however, this has only been confirmed so far for hematological malignancies [46]. Upon treatment with different stimuli, CD56bright NK cells exhibit ability of suppressing proliferation of autologous CD4 T cells via both cytotoxic and immunoregulatory mechanisms, e.g., by secreting the immunosuppressive molecule adenosine (these mechanisms are reviewed in detail in [47]). For some types of solid cancers, in particular lung cancer and breast cancer, the proportion of CD56bright cells in a total amount of tumor-infiltrating NK cells was found to be significantly higher than in the corresponding normal tissues, however, they express low perforin and rather play an immunoregulatory role, but not cytotoxic [48].

### **3.2. Analysis of NK cell subpopulations in peripheral blood lymphocytes of earlystage cervical cancer patients**

Taking into consideration, the proposed model that describes the ability of CD56bright NK cells to circulate among tissues, lymphoid organs, and peripheral blood [45, 48], it can be assumed that altered frequencies of these cells in the blood of cancer patients are highly relevant to immune regulation at the tumor locus. Quantitative assessment of circulating CD56bright NK cell population has been performed for head and neck cancer [49], prostate [50], and breast cancer [51, 52]); we also recently reported our findings concerning circulating NK subsets in women with CIN3 (including carcinoma in situ) and microinvasive carcinoma (stage IA1) of the cervix [23].

Based on the intensity of CD16/CD56 staining, we could distinguish four main subsets of circulating NK cells within CD3-negative lymphocytes (gates P1–P4, **Figure 8**). As expected, we found no significant difference in the frequency of cells within CD16brightCD56dim gate (which encompasses the major pool of circulating cytotoxic NKs), as well as within CD16dim/negCD-56dim and CD16brightCD56neg gates (comprising less abundant populations with poorly established functions) between patients and controls. As opposed to these subsets, a decrease in the frequency of CD16dim/negCD56bright NK cells and, accordingly, higher CD56dim/ CD56bright ratio were observed in cervical cancer patients relative to the control group. We hypothesized this specific alteration reflects a systemic shift in the balance between effector and regulatory NK subsets that occur early in invasive cervical cancer development. One can also speculate this change, along with those described above for M2/M1, neutrophils and MoDC subsets, is part of a complex cervical cancer-related immunoregulatory network. As it is well known that activation of NK cells occurs locally, in our attempt to interpret the obtained data we therefore use the idea that circulating CD56bright NKregs are recruited to the lymphoid tissue (regional lymph nodes) and the primary tumor site, where they are thought to serve as precursors for cytotoxic/effector CD56dim NK cells [45]. This assumption encourages further investigation into the regulatory role of NK cells in cervical cancer progression.

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

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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). 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

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