A significant number of studies have been recently published evidencing about an important role played by the receptors for extracellular ATP and its metabolites as well as enzymes involved in regulating their metabolism in vascular and heart valve calcification (Fish et al., 2013; Towler, 2017; Dou et al., 2018; Golovkin et al., 2018; Kaniewska-Bednarczuk et al., 2018). Previously, it was shown that various receptor isotypes specific to extracellular adenosine (P1 family) and ATP (Р2Х and Р2Y families) were found on smooth muscle cells, the major cell type involved in formation of calcium depositions in the vascular wall (Fish et al., 2013). Nucleotides, in turn, present in extracellular space able to activate P1 and P2 purinergic receptors, are metabolized by a several enzymes acting on ATP and its derivatives. In particular, CD39 (E-NTPDase1—ectonucleoside triphosphate diphosphohydrolase 1) cleaves ATP to two AMPs and two phosphate molecules; the latter is now considered as one of the major cues promoting tissue hydroxyapatite deposition (Fish et al., 2013). Another phosphate molecule is generated by CD73 (Ecto5′NTase—ecto-5′-nucleotidase) converting AMP to adenosine (Fish et al., 2013). Inorganic pyrophosphate (PPi) formed during AMP synthesis mediated by ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) represents a potential inhibitor of hydroxyapatite deposition and subsequent tissue calcification (Fish et al., 2013).

In connection with this, investigating the expression of molecules involved in ATP metabolism during vascular and heart valve calcification potentially underlying its pathogenesis is of high priority.

Furthermore, morphology and biochemical examination of the pig aortic valves demonstrated that CD39 and CD73 were upregulated in both endothelial and interstitial cells (Kaniewska et al., 2014). Culturing endothelial and interstitial cell lines added with nucleotides revealed that CD73 product formation was accelerated in interstitial cells compared with endothelial cells, whereas CD39 activity showed an opposite pattern (Kaniewska et al., 2014).

In addition, CD39 and CD73 are widely expressed by immune cells (Bono et al., 2015; Borg et al., 2017; Zhao et al., 2017a). Purinergic regulation plays a crucial role in T-cell functioning. For instance, ATP is involved in controlling regulatory T-cell differentiation and functional activity. Moreover, low ATP level activates whereas its high amount and prolonged P2X7 stimulation trigger pore formation and T-cell apoptosis (Trabanelli et al., 2012). In turn, adenosine exhibits opposite effects on T cells by suppressing functional activity of some T-cell subsets primarily targeting Th17 cells (Faas et al., 2017) but activating regulatory T cells. Moreover, down-modulated IL-2 production is solely mediated by adenosine resulting in suppressed proliferation and differentiation of naïve T cells towards Th1 and Th2 cells in lymphoid tissues (Csóka et al., 2008). Apart from that, adenosine may block TCR signaling after elevating cAMP level via A2A receptor stimulation (Stagg and Smyth, 2010). Meanwhile, A2A receptor has been identified as the major anti-inflammatory adenosine receptor associated with T cells (Ohta and Sitkovsky, 2001).

Thus, a role played solely by nucleotides as well as purine receptors found in connective tissue cells and lymphocytes egressing from the circulation seems to be important in the pathogenesis of great vessel and heart valve calcification. Moreover, detection of activated T cells in peripheral blood correlating with the level of aortic valve calcification additionally pointed at the close relationship between circulating immune cells and processes occurring in an inflamed aortic tissue (Winchester et al., 2011). In addition, a number of highly differentiated CD3+CD8+ T cells was increased in these patients, whereas a high number of mature CD28-negative cytotoxic T cells was found microscopically in foci of calcification (Winchester et al., 2011).

Thus, the current study was aimed to compare composition of peripheral blood T-cell subsets and assess their surface expression of CD39 and CD73 ectonucleotidases in patients with severe and moderate aortic stenosis (AS) as well as to evaluate involvement of T-cell-mediated immune processes in valve calcification.

The current study was performed with 38 patients suffering from severe calcified aortic stenosis (SAS) undergoing surgical heart valve replacement and 33 patients with moderate AS (MAS) undergoing nonsurgical treatment. Surgical and nonsurgical treatment and follow-up were performed at the Almazov National Medical Research Centre.

All patients underwent comprehensive two-dimensional and Doppler transthoracic echocardiography by using Vivid 7.0 system (GE, USA), according to the current ECHO guidelines. The criteria for severity of aortic valve stenosis included aortic valve area (AVA, cm²) calculated by using continuity equation; AVA indexed for body surface area (AVA/BSA, cm²/m²); and mean transvalvular pressure gradient and peak aortic jet velocity (Vmax).

A multislice spiral computed tomography with Agatston calcium scoring was performed to assess calcium deposits in the heart valves of MAS patients (Falk et al., 2017), reaching 993 (456; 1,968) and 886 (492; 1,229) in males and females, respectively.

Clinical characteristics of patients are presented in Table 1 .

In the comparison group, 30 apparently healthy volunteers (HVs) were enrolled. Due to a limited number of subjects older than 60 lacking signs of any chronic disorder, subjects older than 49 were used as one of the inclusion criteria. Therefore, patients and HVs were significantly age mismatched.

The clinical research protocol was approved by the local Ethics Committee of the Almazov National Medical Research Centre (protocol #83, May 16, 2016) and complies with the Declaration of Helsinki. All patients provided written informed consent.

T cells represent a relatively small cell population infiltrating various tissues. It is known that T cells exhibit an important regulatory effect on inflammatory and reparative processes by releasing signaling cues for macrophages or fibroblasts (Saxena et al., 2014; Hofmann and Frantz, 2015).

CD3+CD4+ and CD3+CD8+ subsets of peripheral blood T cells exhibiting helper and cytotoxic functions, respectively, can be characterized by combining different cell-surface markers, e.g., CD45R0 and CD62L depending on their migration and functional potential. These T-cell subsets are defined as naïve cells (N, CD45R0−CD62L+), central memory cells (CM, CD45R0+CD62L+), effector memory cells (EM, CD45R0+CD62L−), and “terminally differentiated” effector memory cells re-expressing CD45RA antigen (TEMRA, CD45R0−CD62L−). This classification is typically used for CD8+ and CD4+ T cells, although the equivalent TEMRA Th cell subset in healthy donors has not yet been clearly identified due to their low frequency in normal peripheral blood.

After positive and negative selection, naïve CD4+ and CD8+ T cells exit from the thymus into the peripheral blood and express CCR7 and CD62L required for their extravasation through the high endothelial venules into the secondary lymphoid organs, while the presence of CD45RA and lack of CD45R0 indicates that these cell were not encountered by a specific antigen presented by antigen-presenting cells (Sallusto, 2016). Naïve T cells are characterized by highly diverse TCR repertoire encompassing up to 100 million different specificities (Qi et al., 2014), indicating that naïve T cells can respond to various antigens (Kumar et al., 2018). Moreover, in the absence of specific antigen, they also display a high self-renewal potential, but upon specific stimulation in lymphoid tissues, they undergo robust cell expansion followed by differentiation to effector and memory T-cell subsets.

Two major functional T-cell subsets were distinguished (Mahnke et al., 2013) within the CD45RA−CD45R0+ memory T-cell pool: CM T cells, which express lymph node homing molecules CCR7 and CD62L, had very limited effector functions; and CCR7−CD62L− EM T cells are able to migrate to peripheral tissues and exhibit effector functions. Furthermore, CM cells represent a pool of less-differentiated long-living memory cells that recirculate through the secondary lymphoid organs and display high proliferative and renewal capacity. In contrast, EM T cells represent a pool of more-differentiated circulating effector cells that can rapidly enter inflamed tissues due to the upregulated expression of tissue-homing chemokine receptors and adhesion molecules and provide a rapid and effective defense response (Sallusto, 2016). Both CM CD4+ and CD8+ T cells produce high amounts of IL-2 but low levels of other effector cytokines (e.g., IL-4, IL-5, and IFN-γ), whereas EM Th and Tcyt subsets produce a high amount of effector cytokines but a low level of IL-2 (Mahnke et al., 2013).

Compared with naïve and memory T cells, short-lived effector or TEMRA cells display a limited TCR diversity. However, TEMRA cells rapidly migrate to various anatomical sites to promote pathogen clearance by producing inflammatory cytokines and cytotoxicity, yet they exert little proliferative potential antigen-specific stimulation in vitro.

We demonstrated that the percentage of peripheral blood Tcyt was insignificantly increased mainly due to TEMRA Tcyt subset ( Supplementary Table 1 ), whereas frequency of naïve Tcyt and naïve Treg subsets was decreased in patients in SAS patients vs. HVs, respectively ( Figure 2 ). In contrast, in MAS patients vs. HV, the percentage of CM Tcyt subset was significantly elevated (p = 0.01).

Despite few significant differences found in T-cell subset profile observed between patients with moderate aortic stenosis and HVs, it is worth noting about multiple differences between SAS and MAS patients mainly due to opposite changes in similar parameters found in HV group.

It was found that the amount of total Tcyt cells, particularly the relative and absolute numbers of TEMRA Tcyt subset in SAS vs. MAS patients, was considerably increased, which could point most likely at their continuous antigen-specific activation, differentiation, and pro-inflammatory activity. Importantly, no such changes in MAS patients vs. HVs were found.

Mazur et al. performed correlation analyses that demonstrated that mean transvalvular pressure gradient positively correlated with the CD4+CD8+ lymphocyte count and fraction (Mazur et al., 2018). Taking into account the great interest on double-positive T-cell population, we also involved them into the analysis, but having the opportunity to identify this population as heterogeneous, we analyzed it separately as CD4+CD8dim and CD8+CD4dim ( Supplementary Table 1 ). There were no significant changes in absolute and relative count of these populations between MAS and SAS patients and HVs. Meanwhile, positive correlations of CD4+CD8dim and CD8+CD4dim with Agatston calcium score for MAS patients demonstrated their potential interconnection with aortic valve calcification process.

Previously, it was demonstrated that purinergic signaling played an important role in regulating T-cell functions (Ledderose et al., 2016; Golovkin et al., 2018). T cells migrating into the damaged heart tissues upregulate enzymatic machinery for degrading extracellular ATP and NAD to adenosine (Borg et al., 2017).

Numerous studies support the concept that CD39 controls the rate-limiting step in the degradation of extracellular ATP (Eltzschig et al., 2012). While there are multiple AMP sources, the critical bottleneck for the formation of adenosine is CD73 also undergoing upregulated expression (Bonner et al., 2012). Meanwhile, lack of CD73 on T cells enhances tissue fibrosis (Borg et al., 2017). T cells lacking CD73 show accelerated production of pro-inflammatory and profibrotic cytokines (IL-2, IFN-γ, and IL-17) (Borg et al., 2017) presumably accounting for down-modulated CD73 expression on Th and Tcyt subsets in MAS vs. SAS patients vs. HVs.

There are different cellular sources for AMP formation: monocytes and fibroblasts—both largely devoid of CD73—express CD39 and can only degrade ATP to AMP, which may then reach T cells by diffusion (Bonner et al., 2013; Borg et al., 2017).

CD73-derived adenosine acts in an autocrine manner on adenosine receptors A_2aR and A_2bR predominantly expressed by cardiac T cells to counteract pro-inflammatory/antifibrotic cytokines (Borg et al., 2017). For instance, A_2bR stimulation was reported to suppress the release of TNFα from neutrophils and macrophages (Koeppen et al., 2015), inhibit superoxide anion production in neutrophils (Auchampach et al., 2009), augment anti-inflammatory IL-10 production in macrophages (Nemeth et al., 2005), and promote alternative (M2) macrophage activation (Csoka et al., 2012). In addition, T-cell-derived adenosine might influence cytokine release and differentiation of other cardiac immune cells in a paracrine manner, again involving A_2aR and A_2bR (Borg et al., 2017). Meanwhile, CD73 promoting adenosine production is regulated by various cytokines, e.g., interferon (IFN)-β (Kiss et al., 2007), TGFβ, IFN-γ, IL-6, and IL-12 (Regateiro et al., 2011), as well as by paracrine adenosine (Narravula et al., 2000).

While investigating patients with anti-neutrophil cytoplasmic auto-antibody (ANCA)-associated vasculitis (AAV), it was found that on patient T cells, CD39 and CD73 were down-modulated. In CD4+ cells, significant differences in CD73 expression were limited to memory CD45RA− cells, while in CD4+ lymphocytes, differences were significant in both naïve CD45RA+ and memory CD45RA− cells. However, no correlation with disease activity, duration, and ANCA profile was found (Kling et al., 2017).

Previously, it was shown that CD39 and CD73 expression on T cells could be related to the age of human subjects. For instance, CD73 expression on lymphocytes declines with age (Guzman-Flores et al., 2015). Meanwhile, CD39 is more readily induced in CD4 T-cell responses in older individuals (Fang et al., 2016). In our previous investigation with HVs, we found that the number of CD73-expressing naïve T cells was significantly decreased in donors older than 45 compared with younger subjects. However, such pattern was only noted for female subjects (Golovkin et al., 2017). Meanwhile, a significant correlation (r = −0.517, p < 0.05) between CD73 expression on T-cytotoxic cells and male subjects of older age was shown. Besides, female volunteers demonstrated numerous age-related correlations: naïve Tcyt CD73+ (r = −0.520, p < 0.05), CM Tcyt CD73+ (r = −0.397, p < 0.05), naïve Tcyt CD39+ (r = 0.361, p < 0.05), EM Tcyt CD39+ (r = 0.367, p < 0.05), and naïve Th CD39+ (r = 0.378, p < 0.05) (Golovkin et al., 2017). Therefore, CD39 and CD73 expression on T cells may display both age- and sex-related features.

Despite that mean age was lower in HV group, all differences found in our study might not be accounted for only by age-related issues, given especially that the vast majority of them were found between age-matched MAS and SAS patients in terms of T-cell subset composition and percentage of CD39- and CD73-positive cells they contained.

It is known that CD4+CD25+ regulatory T cells (Tregs) are important in maintaining self-tolerance and regulating immune responses in both physiological and pathological settings (Sakaguchi, 2004; Singer et al., 2014; Huang et al., 2015). Tregs demonstrate a great diversity of their composition and functions mediating immune suppression through distinct mechanisms associated with various phenotypical and functional subsets (Caridade et al., 2013). CD39 is a useful marker for identifying CD4+CD25+ Treg cells, even allowing to consider it as a more consistent and reliable marker for Treg cells than CD25 (Deaglio et al., 2007). It was demonstrated that the level of surface CD39 expression on human Tregs parallels that one for intracellular transcription factor FOXP3 (Borsellino et al., 2007; Deaglio et al., 2007). Furthermore, phenotypic analysis comparing CD39+ and CD39− Tregs in adult donors showed that the former expressed a higher level of intracellular FOXP3 and CTLA-4 than did CD39− Tregs (Rissiek et al., 2015). Analyzing chemokine receptor expression revealed that a higher percentage of CD39+ Tregs expressed CCR4, CXCR3, and CCR6 involved in T-cell migration to the site of inflammation, while CCR5 was uniquely expressed by CD39+ Tregs. Upon in vitro activation, proteins GARP and LAP anchored to TGFβ were expressed at a higher level on CD39+ Tregs. Furthermore, it was shown that CD39 plays a non-redundant role in mediating suppressive potential of Treg cells. In addition, FOXP3+CD39+ cells efficiently suppressed CD4+CD25− proliferation in vitro. Moreover, CD39+ vs. CD39− Treg cells displayed a significantly higher suppressive potential (Deaglio et al., 2007).

In contrast, in our study, CD39 was not expressed by total Treg population. Upon that, no significant differences were revealed comparing aortic stenosis patients vs. HV group. However, it is worth mentioning that low- (<20%) and high-percentage (around 50%) Treg subsets tended to emerge, which was observed in all groups. Earlier, increased CD39 expression on Tregs was shown in HIV patients, which was strongly associated with disease progression (Nikolova et al., 2011). Furthermore, an increase in CD39 expression on Tregs has also been detected in tumors and autoimmune diseases (Bastid et al., 2013). Besides, the increased expression of CD39+ Tregs was associated with a poor prognosis for sepsis patients (Huang et al., 2015).

It has been reported that in humans, only 1–5% of circulating FOXP3+ CD4+ T cells express CD73, while its surface expression on human Tregs can be induced upon activation (Sim et al., 2014). In addition, CD73 on Tregs was confirmed to contribute to its functioning, as its suppression limits their suppressive capacity (Zhao et al., 2017b). Besides its enzymatic function, CD73 could be considered as an adhesion molecule that can regulate cell interaction with extracellular matrix components (Zhao et al., 2017b). It seems that a higher CD73+ level on Tregs, particularly CM and naïve subsets, points at stronger suppressive potential in patients with severe aortic stenosis.

A stepwise discriminant analysis uncovered that T-cell subset composition played a pronounced role in distinguishing various groups. Moreover, the expression of the enzymes metabolizing extracellular ATP were of strongest significance particularly involving T cells exhibiting phenotype CD73−CD39−, CD73+CD39−, and CD73−CD39+. Thus, parameters of T-cell-mediated immunity and purinergic regulation were substantially different in SAS vs. MAS patients as well as in HV group.

Moreover, discriminant analysis uncovered that patients with various stenosis magnitude formed rather separate groups, and major parameters were sufficient for referring each patient to either group. At the same time, the minimum differences between MAS patients and HV group in parameters noted above were found, which was not observed by comparing SAS patients vs. HV group. It seems that this discrepancy was due to the intensity of aortic calcinosis found in both patient groups.

Examining interstitial cells isolated from aortic valve biopsies demonstrated that the majority of them displayed CD39−CD73− phenotype. Upon that, further passages were shown to increase proportion of the cells expressing these markers up to 100%. Besides, our immunohistochemical data demonstrate that despite the presence of CD3-positive cells in the calcified aortic valves tissue, the number of CD39-positive cells is low, and CD73-positive cells are almost absent. Previously, it was shown that the low or even insufficient level of CD39 and CD73 expression was observed on the cells from calcified vs. non-calcified aortic valves (Kaniewska-Bednarczuk et al., 2018), which was accompanied with reduced activity of NTPDase and e5NT by 35% and 50% (Kaniewska-Bednarczuk et al., 2018), respectively, in calcified lesions. In our study, it was noted that stimulation with either ATP or adenosine postponed (for 7 days) upregulated CD73 expression detected on interstitial cells, whereas the percentage of CD39−CD73+ cells was even decreased 24 h after the onset of experiment. Thus, the data obtained in our study confirm a hypothesis (Kaniewska-Bednarczuk et al., 2018) proposing a down-regulated expression and decreased activity of ectoenzymes CD39 and CD73 on the cells from the calcified aortic valves. Despite other data contradicting those of our study, we found higher CD73 expression and activity level for the calcified vs. non-calcified valve cells (Mahmut et al., 2015). Moreover, we also demonstrated that ectonucleotidase expression level did not differ after a short-term stimulation with either adenosine or ATP, perhaps, on the one hand, pointing at the potential role for purinergic signaling in aortic valve calcification and, on the other hand, maybe implying lack of plasticity in enzymatic system under pathological settings that make it unresponsive to the targeted activation. Confirmation of the latter can be considered by the lack of changes in the expression of genes associated with proosteogenic activity.

Cell functions regulated by purinergic signaling seem to be of importance in various pathological conditions including formation of aortic stenosis and its calcification, which may be related to activity of peripheral blood T-cell subsets and aortic valve interstitial cells. It was found that patients with severe calcified aortic stenosis contained higher percentage of CD73+ Th cells primarily due to CD73+ naïve as well as naïve and CM Treg cell subsets. Along with that, frequency of the same CD73+ T-cell subsets in patients with moderate aortic stenosis was decreased, thereby confirming a concept that the lack or decreased CD73 T-cell expression enhances production of pro-inflammatory and profibrotic cytokines exhibiting overall pro-inflammatory effects. Strikingly, no significant differences in CD39 expression level were found in MAS and SAS patients compared with HV group. Moreover, in our study, a significance of the surface ectonuclease expression on aortic interstitial cells was also demonstrated. In particular, such cells isolated from aortic calcified valve samples turned out to mainly display CD39−CD73− phenotype, which increased percentage of CD73+ cells in response to stimulation with adenosine or ATP. Overall, the data obtained demonstrated that purinergic signaling was involved in the pathogenesis of aortic stenosis and calcification potentially acting via various cell types (T cells and aortic interstitial cells), wherein among enzymes, degrading extracellular ATP CD73 rather than CD39 played a prominent role.

The clinical research protocol was approved by the local Ethics Committee of the Almazov Federal Medical Research Centre (protocol #83, May 16, 2016), and complies with the Declaration of Helsinki. All patients provided written informed consent.

KI and SM performed flow cytometry research. ZE, MP, TV, and IO created the database of patients and healthy volunteers. MA, SA, and SD performed valve interstitial cells research. GA, KI, and MO designed the research. ML performed all histological and immunohistochemical analyses. GA and KI analyzed the data. GA, KI, and ID wrote the paper.

Clinical studies were performed at the Almazov National Medical Research Centre and supported by the Russian Foundation for Basic Research (project 18-015-00016). Laboratory studies were financially supported by the Russian Science Foundation (grant/award no. 19-75-20076).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.