Regulatory CD4/CD8 lymphocytes
The population of CD4 Tregs was determined by analyzing two combinations of essential Treg-markers: CD4+CD25+/highCD127low/neg and CD4+CD25+FoxP3+ [25]. Total CD25+, FoxP3+, CD4+CD25+, CD4+CD25high, and CD4+FoxP3+ lymphocytes were also enumerated. No significant differences in the numbers of these cell populations were found between age subgroups of healthy controls. Patients with CIN3/cancer in situ and CC stage IA showed higher frequencies of CD4+CD25+ and CD4+CD25high lymphocytes compared to the controls (Fig.1); the level of CD25 marker expression in total lymphocyte population was also increased in patients (p < 0.001, data not shown). Similarly, higher median levels of CD4+FoxP3+ cell numbers could be observed in patient groups than in the controls (with this result proved to be statistically significant for CC stage IA patients, p = 0.0006, Fig.1), and increase in the total number of FoxP3-expressing lymphocytes was also detected (p < 0.05, data not shown). The proportion of CD4+CD25+FoxP3+ cells among CD4-positive lymphocytes or total lymphocyte population was significantly elevated in peripheral blood samples from patients with CIN3/cancer in situ and CC stage IA (p = 0.037 and p = 0.0004, respectively), whereas the difference in the number of cells defined as CD4+CD25+/highCD127low/neg lymphocytes between the groups of patients and controls appeared less pronounced. The percentage of CD4+CD25+/highCD127low/neg lymphocytes was moderately correlated with the percentage of CD4+CD25+FoxP3+ lymphocytes (r = 0.604, p < 0.01), probably due to higher variability in CD127 expression in Tregs, as explained by Santegoets and colleagues who showed that FoxP3-expressing cells comprised approximately 70–85% of CD25+CD127low population [25]. In addition to increased frequencies of peripheral CD4 lymphocytes with Treg-related phenotype, we revealed a higher concentration of serum TGFβ1 in patients with preinvasive and microinvasive CC (p < 0.001 for both patient groups, Fig.2a). Moderately strong positive relationship was found between serum TGFβ1 level and the percentage of CD4+CD25high lymphocytes (r = 0.552, p < 0.001, Fig.2b).
The ratio of circulating CD8+ Т-cells to CD4+CD25+FoxP3+ regulatory lymphocytes (CD3+CD8+/Treg, Fig.3) was lowered in patients relative to the controls (p = 0.002 for the total patient group) as a result of increasing frequency of regulatory cells and declining numbers of CD3+CD8+ cells that could be seen upon invasive cancer formation (at the same time, there was no statistically significant difference in the number of CD3+CD4+ Т-cells between the patient and the control groups).
Regarding CD8 Tregs, there is currently no consensus on a set of phenotypic markers that defines this population of lymphocytes possessing immunosuppressive properties. Nevertheless, it is known that, like CD4 Tregs, they are characterized by constitutive CD25 expression and, in contrast to effector CD8 T cells, they are positive for FoxP3. As shown in Fig.4, in blood samples from CIN3/cancer in situ/CC stage IA patients, the mean percentage of CD8+CD25+ lymphocytes was increased compared to control samples (p = 0.018); at the same time, a relatively wide range of values could be detected in healthy controls, which is in line with findings from others [26]. As confirmed by ex vivo testing, the CD8+CD25+FoxP3+ phenotype describes the population of CD8 cells with immunosuppressive capacity [26]. In our study, the frequency of CD25+FoxP3+ cells among circulating CD8+ lymphocytes was higher in CIN3/cancer in situ and CC stage IA samples compared to the controls (p = 0.0009 and p = 0,007, respectively, Fig.4). The average proportion of CD8+CD25+FoxP3+ cells in the whole lymphocyte population was also higher in cancer patients than in control subjects, but the difference was less pronounced (p = 0.002 for CIN3/cancer in situ and p = 0.017 for CC stage IA), presumably due to reduction in the level of CD8-bearing lymphocytes observed in women with cancer and low abundance of this cell population (0.1% on average) among peripheral blood lymphocytes from both patients and controls. In addition, it has been described that low level (or absence) of CD127 expression is characteristic of CD8 Tregs, therefore we analyzed CD8+CD25+CD127low/neg lymphocytes and found their relative amount calculated as percentage of CD8+ lymphocytes was increased in patient-derived peripheral blood (similar to CD8+CD25+FoxP3+ counts) at p < 0.05, however, the change in the frequency of these cells expressed as a percentage of total lymphocytes was not statistically confirmed (Fig.4). Analysis revealed moderate correlation (r = 0.539, p < 0.001) between the percentages of CD4+ и CD8+ Tregs in patients (while these parameters were not correlated in healthy donor-derived blood samples, see Figs. 9 and 10), suggesting that coordinated expansion of immunosuppressive cell populations takes place upon disease progression. Taken together, these observations support the notion that early stages of CC development and progression (including, pre-invasive conditions) can be accompanied by a systemic increase in the frequencies of both CD4+ and CD8+ cells displaying Treg-associated phenotype.
Expression of CD95 in CD4+ and CD8+ T cell populations
CD95 membrane expression was estimated by flow cytometry both in total T-cell population and its CD4+/CD8+ subsets (Fig. 5). No significant differences in the level of CD95 expression on T cells, as well as in the number of CD4+ and CD8+ T cells could be detected between age subgroups within the control group. In patients, the amount of CD3+CD95+ lymphocytes (gate P1, Fig. 5) was increased as compared to the control group (p = 0.043), with this increase being more pronounced (p = 0.008) in a subpopulation of T cells expressing intermediate/high level of surface CD95 (gate P2). In general, the abundance of CD95+ cells among the total population of circulating lymphocytes was higher in patients with CIN3/cancer in situ and CC stage IA (p = 0.01, data not shown). Separate assessment of CD4+ and CD8+ T cells revealed different trends of CD95 expression during CC development: the proportion of CD4+CD95+ cells among T lymphocytes was higher in blood samples taken from patients (p < 0.05; gates P3, P4). On the contrary, a trend towards decreased proportion of CD8+CD95+ T cells was observed (p > 0.05) upon invasive cancer establishment (gates P5, P6). The number of CD95-negative cells was diminished within both CD4+ and CD8+ T cell populations of cancer patients, but in the case of CD8+ cells this reduction was more significant (p = 0.01, data not shown). Consequently, the ratio of CD95-positive T helpers to CD95-negative demonstrated significant increase upon disease progression (p = 0.005), whereas killer T cells exhibited no evident change in CD95+/CD95− ratio (Fig. 5). Altogether, we can conclude that an increase in the frequency of circulating CD95+ Т cells seen in CC patients was associated primarily with the CD4+ T-cell population (with comparable, as mentioned above, frequencies of blood CD4+ T cells in CC patients and controls).
The level of apoptosis in peripheral T lymphocytes
We further explored whether the expansion of suppressive Tregs, along with elevated levels of secreted TGFβ1 and CD95 death receptor surface expression, could be accompanied by enhanced level of T cell apoptosis in circulation of CIN/CC patients. The amount of apoptotic lymphocytes was evaluated by Annexin V (Anx) binding; dead cells and cellular debris were identified based on FSC/SSC parameters combined with propidium iodide (PI) staining, and were excluded from analysis. The quantity of Anx+PI+ lymphocytes (late apoptotic or necrotic cells) was relatively constant among peripheral blood samples from all groups examined (1.73 ± 0.20% for the control group and 1.93 ± 0.20% for the patient group, p > 0.05, data not shown), so these cells were also excluded.
As shown in Fig.6, the mean percentage of Т lymphocytes that bound Annexin V was higher in patients with CIN3/cancer in situ/ CC stage IA than in the controls at p < 0.01 (gates P1, P2). Further, by performing three-color staining, we assessed the abundance of CD3+Anx+ T cells co-expressing CD95 that could render them more susceptible to extrinsic/death receptor-mediated apoptosis. Indeed, there was a statistically significant increase in the proportion of CD3+Anx+CD95+ cells among CD3+ T lymphocytes in blood samples obtained from CIN/CC patients relative to the control samples (p < 0.01, gates P3, P4, Fig.6). In comparison, the amount of CD3+Anx+ cells, negative for CD95 expression, constituted a much smaller proportion of T lymphocytes (around 4 times less than the amount of CD3+Anx+CD95+ cells, in both patients and normal controls) and showed no significant difference between the groups examined (2.26 ± 0.28% for healthy controls and 3.00 ± 0.58% for patients, p > 0.05, data not shown). Consequently, the prevalence of CD3+Anx+CD95+ cells in population of Т lymphocytes calculated as CD3+Anx+[CD95+/CD95−] ratio (Fig.6) was higher in peripheral blood of patients (p = 0.051); furthermore, the percent of CD3+Anx+CD95+ cells appeared to be strongly correlated with the percent of CD3+Anx+ cells (r = 0.937, p < 0.001), suggesting growing importance of CD95-dependent pathway in apoptosis of peripheral Т lymphocytes during the development of invasive CC. As for CD3+AnxnegCD95+ cells, which constitute a major population of T lymphocytes and can be defined as activated effector T cells, the difference between healthy donors and CIN/CC patients was not statistically significant, despite some weak trend towards higher percentage of these cells in patients (55.2 ± 2.5% and 51.8 ± 2.0% of T cells in patients and controls, respectively; data not shown). Together, these findings support an assumption that the development of invasive CC may be associated not only with up-regulation of CD95 expression in peripheral blood lymphocytes, but rather with exacerbation of apoptosis-related processes in CD95+ T cells. These results also agree with our previous study demonstrated enhanced activity of the main CD95-regulated caspases (specifically, caspases −8 and −3) in total fraction of peripheral blood mononuclear cells at such early stages of CC progression as intraepithelial and microinvasive carcinoma [27].
In an assessment of the entire population of blood lymphocytes, an increase in the number of CD95+ cells co-stained with Annexin V was also noticed in cancer patients (p < 0.05; Fig. 7). This increase might be due in part to various populations of non-T lymphocytes (for example, B cells and natural killer cells) as we detected higher percent of CD3−Anx+ cells in blood from patients (6.74 ± 0.54% of lymphocytes) compared to normal controls (5.23 ± 0.43%, p < 0.05). However, when assessing the total lymphocyte fraction, the difference in the ratio of %Anx+CD95+ to %Anx+CD95− cells between patients and controls was not found to be statistically significant (Fig. 7). These data seem to indicate, again, the specificity of alterations in apoptotic processes triggered by external signals (such as CD95L) with respect to T cell population during CC development, although the possibility that activation of extrinsic apoptotic pathways (including CD95-mediated) occur in other types of lymphocytes should not be discarded. Further studies are needed to corroborate this notion; in addition, the above observations of different pattern of changes in CD95 expression in the two major T cell subsets (CD4+ and CD8+) may imply different intensity of apoptotic processes, an issue which is also to be further investigated using modified flow cytometry staining/gating strategy.
The innate arm of immunity: Regulatory NK lymphocytes (NKregs)
NKregs have been described as a minor subpopulation of circulating natural killer cells having CD3negCD16dim/negCD56bright phenotype. Compared to CD16+CD56dim NK cells which represent the majority of NKs in peripheral blood, they are associated with relatively low cytolytic capacity (in the absence of appropriate activating signals), but are known for their high ability to produce cytokines and chemokines (including IFNγ, TNFα, GM-CSF, IL-10, IL-13, CCL3, CCL4), and therefore they are considered as immunoregulatory NK subset, by analogy with regulatory T cells [28]. In our study, patients showed lower frequencies of CD16dim/negCD56bright cells compared to controls (p = 0.027, Fig. 8), while no differences in percentages of other existing NK cell subtypes (i.e. CD16brightCD56dim, which is the major circulating subset, and two minor subsets of CD16dim/negCD56dim and CD16brightCD56neg cells [29]) were revealed (data not shown). Correspondingly, the ratio of %CD56dim NKs to %CD56bright NKs displayed a tendency to increase in cervical cancer patients relative to the controls (significantly at p = 0.016 in stage IA patient group, Fig.8), presumably indicating a systemic change in the balance between effector and regulatory cells within the innate immune system promoted by invasive cervical cancer development. Along with Treg expansion, this change may contribute to systemic immune suppression in CIN/CC patients.
Analysis of correlations: Putative mechanisms for the establishment of a suppressed immune status of cervical carcinoma patients
A total of 80 combinations of phenotypic parameters (gates) analyzed in the present study were nominally split into 3 functional categories (“Regulatory cells”, “CD95-expressing T cells”, and “Apoptosis”), and a search for correlations between them was performed on the basis of flow cytometric data. Most significant relationships (r > 0.6, p < 0.05) existing between the parameters (i.e. lymphocyte subpopulations) that were assigned to these functionally distinct categories are summarized in Figs.9 and 10. As follows from the results obtained in the whole cohort of participants (controls + patients), an increase in the percentage of CD95-positive CD4 or CD8 T cells relative to corresponding CD95-negative populations was correlated with the increased proportion of apoptotic (Anx+) lymphocytes co-expressing CD95, with this correlation being more pronounced in CD4+ Т cells, than in CD8+ T subset (Fig.9). Concomitantly, the lack of CD95 expression on CD4+/CD8+ T cells was associated with the absence of Annexin V binding in total T lymphocytes (as determined in independent probes). Interestingly, in contrast to CD4+ T-cells, the increase of CD95+ to CD95− ratio in CD8+ Т cell population strongly correlated with the percentage of CD3+CD95+ Т lymphocytes that were negative for staining with Annexin V, as well as with the total amount (%) of CD3+CD95+ cells. In sum, the in vivo existence of these relationships could be accounted for based on the concept that upregulation of CD95/Fas receptor surface expression (resulting, for example, from lymphocyte activation in response to infection) represents a critical mechanism of induction of apoptosis in Т lymphocytes upon interaction with death ligands and, along with this, the observed change in CD95 may have distinct functional significance for CD4+ and CD8+ T cell subsets. In addition to these results, moderately strong positive correlation was revealed between the frequencies of cells expressing Treg-associated phenotype and CD4+CD95+ T lymphocytes (Fig. 9).
We next hypothesized that coordinated changes in the frequencies of the different populations of lymphocytes upon disease development may imply involvement of these populations in a common mechanism exploited by cervical cancer to establish immunosuppression. Therefore similar correlation analysis was done separately for the group of healthy women and the group of women with cervical neoplasms (CIN3/cancer in situ/ CC stage IA) in order to find out which of the relationships emerge (or become significantly stronger) in cancer, but are absent (or negligibly weak, r < 0.6) in normal controls. The scheme in Fig. 10 shows correlations between the proportions of lymphocyte populations that were found to have an absolute value >0.6 in the patient group (at p < 0.05), but <0.6 in the control group. In comparison with the results obtained using the full sample, CIN/CC patients exhibited stronger correlation between the expression of CD95 on CD4+ T cells and the frequency of regulatory CD4+CD25+/high and CD4+FoxP3+ lymphocytes. The data also showed a strong negative correlation between the frequency of CD4+CD25+/high cells (as well as overall expression of CD25 marker in circulating lymphocytes) and CD3+CD8+/Treg ratio in women with a diagnosis of CIN3/cancer in situ/ CC stage IA, but not in healthy donors. Additionally, the lymphocyte expression of FoxP3 factor and the level of CD95-associated apoptosis in T cells (estimated as the proportion of CD3+CD95+Anx+ lymphocytes) were correlated only in patient group (Fig. 10).
When considering the relation between the level of apoptosis and differential expression of CD95 in CD4+ and CD8+ T cells, one can notice that it is within the CD8+ T cell subset where the correlation between the relative amount of CD95-expressing cells (CD3+CD8+[CD95+/CD95−] ratio) and the level of Anx binding with CD95+ lymphocytes was found in the CIN/CC patient group only, whereas for CD4+ T lymphocytes this correlation was detectable in the control group as well (r > 0.7). Likewise, a correlation between elevated percentage of total CD95-expressing T lymphocytes (%CD3+CD95+/high) and elevated proportion of CD95-expressing cells within CD4+ subset (CD3+CD4+[CD95+/CD95−] ratio) was observed in patients (in the case of CD8+ T-cell subset, the correlation between these parameters had r > 0.6 both in patients and controls).
For each phenotypic parameter measured by flow cytometry, we also analyzed the empirical distribution and determined 0.1 and 0.9 quantiles based on the values obtained for control group. The [0.1; 0.9] interquantile interval was taken as the ‘normal’ range and the number of values lying outside this interval in the patient group was divided to the total number of observations to obtain the proportion of values that differ from the normal range (Fig. 11). Reasoning from the results, we may hypothesize that Tregs may be one of the primary sources for other changes in cellular immune parameters, as more than 40–50% of patients with CIN3 or initial stages of CC progression (0-IA1) showed higher frequencies of Treg cells (both within CD4 and CD8 subset) in circulation in comparison with the ‘normal’ range. About one third of cases exhibited altered CD95 expression on T cells (above the ‘norm’ for the CD4+ subset and below for CD8+) and increased levels of Annexin V binding (Fig. 11). Taken together, women with early neoplastic lesions of the cervix, such as carcinoma in situ and microinvasive carcinoma, displayed a coordinated increase in expression of markers of regulatory T cells in blood CD4+/CD8+ lymphocytes, along with more pronounced cross-relationships between Treg numbers, CD95 expression on CD4+/CD8+ T cells, and apoptosis, with these interrelated changes being suggestive of the mechanisms responsible for the development of systemic immunological deficiency throughout cervical cancer progression.