Regulatory T-lymphocytes (Tregs) play a critical role in maintaining proper immune system function. They control the activation and proliferation of other immune cells, preventing autoimmune and pathological reactions [1, 2]. The majority of Tregs are CD4⁺ T-cells, which can be identified by high CD25 (interleukin (IL)-2R$\alpha$), low CD127 (IL-7R$\alpha$), and active forkhead box P3 (FOXP3) transcription factor synthesis [3]. Like other CD4⁺ T-cells, Tregs are susceptible to Human Immunodeficiency Virus (HIV)-infection, and their absolute counts decrease during disease progression [4, 5, 6, 7]. However, studies show that in people living with HIV (PLWH), Tregs largely maintain their functional activity despite the decline in numbers and can even upregulate pathways associated with a more suppressive (activated) phenotype [8, 9, 10, 11, 12].

The dynamic interplay between Tregs and HIV-infection is evident in a subset of patients known as immunological non-responders (INRs). INRs are PLWH whose viral replication is controlled by antiretroviral therapy (ART), yet their CD4⁺ T-cell counts remain low. Compared to immunological responders (IRs) who show increased CD4⁺ T-cell counts after ART, INRs face higher morbidity and mortality risks [13, 14]. Studies report INRs have increased Treg frequencies despite low absolute Treg counts [15, 16, 17]. Possible explanations include: (1) The need to prevent chronic immune activation caused by microbial translocation [18, 19]. (2) The unknown underlying pathogenic mechanism in which increased Treg frequencies indicate disease progression [20, 21, 22]. (3) Lesser Treg susceptibility to productive viral infection compared to other CD4⁺ T-cell subsets [23, 24, 25]. Furthermore, almost nothing is known on the Treg functional activity in INRs. Studies show that the expression of genes specific to Treg function and those upregulated by the master transcription factor FOXP3 was low in INRs [26]. Thus, data on Tregs in INRs are little or contradictory, and the mechanisms behind Treg dynamics remain unclear.

Chronic lymphopenia is a key feature of INR. In a lymphocyte-deficient setting, remaining naïve and effector/memory T-cells rapidly proliferate [27, 28] due to interactions between the T-cell receptor on CD4⁺ T-cells and low-affinity self-peptides presented by major histocompatibility complex, potentially leading to autoimmune diseases [29, 30, 31, 32]. To manage intense cell division, additional Tregs may be generated. Indeed, lymphocyte regeneration produces both effectors and Tregs [33, 34], with the latter forming from cycling conventional CD4⁺ T-lymphocytes [34, 35, 36]. These peripherally-derived Tregs can develop independently of the thymus and resemble thymus-derived Tregs in phenotype and function [35, 37, 38]. However, the quantity and function of Tregs in chronic lymphopenia are not well-studied.

This study aimed to examine Treg dysregulation in immunological non-responders to ART.

The study received approval from the Perm Krai Center for the Prevention and Control of AIDS and Infectious Diseases Institutional Review Board (IRB00008964). Written informed consent was obtained from all participants. Three groups were assessed (Table 1):

(1) Immunological non-responders to ART (INR; peripheral CD4⁺ T-cells below 350/µL; n = 40);

(2) Immunological responders to ART (IR; peripheral CD4⁺ T-cells above 350/µL; n = 42);

(3) Healthy control group (HC; uninfected volunteers; n = 23).

All PLWH included in the study had been on ART for over two years. Typical treatment regimens usually consisted of 2 nucleoside reverse transcriptase inhibitors with ritonavir-boosted protease inhibitor or non-nucleoside reverse transcriptase inhibitor. Patients exposed to interferon or direct acting antiviral therapies for hepatitis C virus (HCV)-infection were excluded from the study. All study participants were screened and confirmed to be free of hepatitis B virus or tuberculosis. Former injection drug users reported no recent drug use, which was corroborated by negative results on urine lateral flow immunochromatographic assays («ИХА-5-МУЛЬТИ-ФАКТОР», Factor-MED, Moscow, Russia).

From each participant, approximately 50 mL of blood were collected into tubes pre-coated with ethylenediaminetetraacetic acid (Guangzhou Improve Medical Instruments Co, Ltd., Guangzhou, China). Plasma HIV levels were assessed with a PCR-based technique using the Versant HIV-1 RNA 3.0 assay b kit (cat.# 118276, Bayer Corporation, Tarrytown, NY, USA) according to manufacturers’ instructions.

Peripheral blood mononuclear cells (PBMCs) were isolated through density gradient centrifugation using Diacoll-1077 (cat.# Diacoll 1077, Moscow, Dia-M, Russia), cryopreserved in heat-inactivated fetal bovine serum (cat.# A5670801, Gibco, South America) supplemented with dimethyl sulfoxide (cat.# A7248, AppliChem, Germany), and subsequently stored in liquid nitrogen. Thawed PBMCs were analyzed with a BD LSRFortessa flow cytometer (B6-R4-V6-UV2, BD Biosciences, NJ, USA). Monoclonal antibodies for cell phenotyping included: anti-CD25-BUV395 (cat.# 564034, Becton Dickinson, NJ, USA), anti-CD127-BV786 (cat.# 563324, Becton Dickinson), anti-CD71-BV42 1(cat.# 562995, Becton Dickinson), anti-CCR7-PE-Cy7 (cat.# 557648, Becton Dickinson), anti-CD3-AF700 (cat.# 557943; Becton Dickinson), anti-CD45RA-BV650 (cat.# 304135; Biolegend, CA, USA), anti-CD4-Qdot605 (cat.# Q10008; Invitrogen, CA, USA), anti-FOXP3-PE (cat.# 12-4777-42; eBioscience, MA, USA). Cell viability was evalusted using the LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (cat.# L34966; Invitrogen). Antibody titration was performed to optimize staining resolution. Cell subsets were gated using Fluorescence Minus One (FMO) controls.

CD4⁺ T-cell subsets were assessed according to [39]: naive (CD45RA⁺CCR7⁺); central memory (CD45RA^–CCR7⁺); effector memory (CD45RA^–CCR7^–), and effector memory cells re-expressing CD45RA (TEMRA; CD45RA⁺CCR7^–). Tregs were identified as CD25⁺CD127^loFOXP3⁺ CD4⁺ T-cells. Conventional T-lymphocytes (Tconvs) were determined as CD25^–/loFOXP3^– CD4⁺ T-cells. Activated/cycling lymphocytes were identified as cells positive for transferrin receptor 1 (CD71) which correlated with Ki-67 expression [26].

MitoTracker Green (cat.# M7514, Thermo Fisher Scientific, MA, USA) and MitoTracker Orange dyes (cat.# M7510, Thermo Fisher Scientific) were used to quantify mitochondrial mass and transmembrane potential in CD4⁺ T-cells. Mitochondrial adaptation to transition from quiescent to activated/cycling state was analyzed by calculating shifts in median fluorescence intensities ($\mathrm{\Delta }$MFI, %) using data from activated/cycling (CD71⁺) and quiescent (CD71^–) naïve (CD45RA⁺) or memory (CD45RA^–) regulatory and conventional CD4⁺ T-cells by the following formula:

(MFI_{activated/cycling cells} * 100 % / MFI_{quiescent cells}) – 100 %

For the suppression assay, CD4⁺ T-lymphocytes and Tregs were isolated from freshly prepared PBMCs using the Dynabeads Regulatory CD4⁺/CD25⁺ T Cell Kit (cat.# 11363D, Invitrogen) according to the manufacturer’s protocol. Briefly, PBMCs were consistently incubated with the antibody mix and depletion Dynabeads for the negative selection of CD4⁺ T-cells with a DynaMag-5 magnet (cat.# 12303D, Life Technologies, UK). Isolated cells were incubated with Dynabeads CD25 and placed on a magnet to separate CD25^–cells and CD25⁺ Tregs. The latter were then incubated with a DETACHaBEAD release reagent to get rid of CD25-binding beads. The purity of CD4⁺CD25^– and CD4⁺CD25⁺ T-cells was determined via flow cytometric analysis utilizing a CytoFLEX S instrument (B5-R3-V3-NUV2, Beckman Coulter, CA, USA).

Isolated cells were resuspended in a complete medium [RPMI-1640 supplemented with glutamine and HEPES (cat.# SLM-140-B, Sigma, MA, USA), 10% heat-inactivated fetal bovine serum (cat.# A5670801, Gibco), and 1% penicillin/streptomycin (cat.# p4333, Sigma )] at 37 °C under 5% CO₂. CD25^–CD4⁺ T-cells were either cultured alone or co-cultured with autologous Treg cells at a 1:1 ratio in 96-well U-bottom plates (cat.# 07-200-95, Costar, MA, USA) with a total volume of 200 µL. All cells were treated with phytohemagglutinin (15 µg/mL; cat.# 27651, Serva, Germany). Twenty-four hours later, cells were harvested and dyed with anti-CD3-APC (cat.# 300412, Biolegend), anti-CD4-AF700 (cat.# 344622, Biolegend), and anti-CD69-FITC (cat.# 310904, Biolegend) antibodies. The suppressive activity of Treg cells was quantified using as an index of suppression calculated with the following formula: 1-(frequency of CD69⁺ cells among PHA-treated lymphocytes in the presence of Tregs/frequency of CD69⁺ cells among phytohemagglutinin (PHA)-treated lymphocytes in the absence of Tregs).

The data were reported as medians and interquartile ranges (25th to 75th percentiles). The statistical analysis and data visualization were performed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, CA, USA). The Kruskal-Wallis test compared three groups, with Dunn’s test for multiple comparisons. The Mann-Whitney U-test compared two groups. The Wilcoxon matched-pairs signed rank test compared paired data. Correlation analysis used Spearman’s method.

In PLWH, the proportion of CD71⁺ cells among CD4⁺ T-lymphocytes was significantly elevated (Fig. 1A, Supplementary Fig. 1A). This indicates high immune activation and cell cycling. The highest frequency of activated/cycling CD4⁺ T-cells was found in the INR group, with immunological non-responders having 3.1 times more activated/cycling CD4⁺ T-cells compared to healthy controls, and 1.7 times more compared to immunological responders.

We examined activated/cycling cell frequencies among conventional and regulatory CD4⁺ T-lymphocytes (Supplementary Fig. 1B). Tregs had more activated/cycling cells than Tconvs (Fig. 1B). There was a direct correlation between Treg and Tconv cycling rates across all groups (Fig. 1C). These findings suggest Tregs control Tconv proliferation in normal and lymphopenic settings [34, 40]. Notably, the highest frequencies of activated/cycling Tregs among CD4⁺ T-cells were observed in INRs. Nevertheless, the abundance of activated Tregs does not appear to effectively control the proliferation of Tconv cells. This suggests increased Tconv turnover in INRs may result from a disrupted Tconv/Treg ratio, Treg dysfunction, or both.

Analysis of FOXP3 expression in CD4⁺ T-cells showed that CD25⁺CD127^loFOXP3⁺ lymphocytes upregulate FOXP3 when entering the cell cycle. This pattern was observed across all study groups, including immunological non-responders (MFI in CD71^– and CD71⁺ Tregs: 1134 vs. 2152; p 0.001), immunological responders (1088 vs. 2162; p 0.001), and healthy controls (737 vs. 1526; p 0.001). These findings suggest that the activation and proliferation of Treg phenotype cells is accompanied by enhanced FOXP3 expression, the key transcription factor defining suppressive function. Thus, activated/cycling Treg phenotype lymphocytes may be considered bona fide Tregs.

We found a higher frequency of Tregs among total CD4⁺ T-cells in immunological non-responders compared to healthy controls. Treg frequency was 8.0% (6.7–9.3%) in INRs, 7.5% (5.7–8.4%) in IRs, and 6.3% (5.0–7.7%) in HCs (p 0.01 for INR vs. HC). To account for CD4⁺ T-cell heterogeneity, we analyzed Treg distribution across CD4⁺ T-cell subsets with varying maturation states: naive, central memory, effector memory, and TEMRA cells, further subdivided by CD71 expression. The pattern observed in the total CD4⁺ T-cell pool was only found among quiescent effector memory cells (Table 2).

The analysis of the activated/cycling cell population (Supplementary Fig. 2) revealed a distinct pattern. Treg frequencies were lower in immunological non-responders compared to healthy controls, specifically among naive and central memory T-lymphocytes (Fig. 2). The ratio of conventional to Tregs, a commonly used metric to assess Treg suppressive function in vitro, showed an excess of conventional CD4⁺ T-lymphocytes relative to Tregs, supporting Treg deficiency in these subsets (data not shown).

The functional state of activated/cycling Tregs was assessed by measuring the expression of molecules involved in their suppressor activity (Supplementary Fig. 3). Memory suppressor cells showed higher FOXP3 levels than naive Tregs in all groups (Fig. 3A). FOXP3 in naive Tregs didn’t differ between immunological non-responders, immunological responders, and healthy controls. PLWH had higher FOXP3 in memory Tregs than healthy individuals. A similar pattern was seen for CD25 on activated/cycling Tregs (Fig. 3B). Naive Tregs had lower CD25 than memory suppressor cells. INRs and IRs didn’t differ in CD25 on naive cells but had higher levels than HCs. In activated/cycling memory Tregs, INR, IR, and HC had similar CD25 levels, with IRs being slightly higher. These results suggest activated/cycling Tregs from INRs aren’t deficient in FOXP3 or CD25, crucial for suppressor function.

To assess Treg functionality, a suppression assay was performed. The results showed that Tregs received from INRs were able to effectively suppress the activation of stimulated CD4⁺ T cells (Fig. 4). These in vitro findings indicate no apparent defects in Treg functionality in INRs when suppressor cells are present in excess (1:1 ratio). However, it is important to note that potential issues with activated/cycling Tregs may have been masked by the presence of quiescent Tregs in this assay.

Mitochondrial activity is crucial for cell division [41]. After observing high turnover of Tregs in INRs, we assessed the mitochondrial function of these cells. We measured mitochondrial mass and transmembrane potential in naive and memory Tregs across the three groups (Supplementary Fig. 4). Our results showed no differences in these parameters between activated/cycling Tregs from INRs, IRs, and HCs (Table 3).

Given cell division’s energy demands, we assessed mitochondrial condition by measuring shifts in mitochondrial mass and transmembrane potential as cells moved from a quiescent to an activated/cycling state. Naive Tregs significantly increased these parameters when cycling (Fig. 5A). There was a gap of over 15 % in both metrics between INRs and healthy subjects, but it wasn’t statistically significant. In contrast, memory Tregs lost some mitochondrial mass and charge when cycling (Fig. 5B). All clinical groups showed similar decreases in Treg mitochondrial mass, but changes in transmembrane potential varied. Healthy controls maintained transmembrane potential, but PLWH, especially INRs, showed a decrease of up to 23.4 %. Mitochondrial depolarization, as seen in INRs, is significant for inducing apoptosis and cell cycle arrest [42, 43, 44, 45].

Notably, activated/cycling conventional CD4⁺ T-lymphocytes showed similar changes in mitochondrial mass and transmembrane potential as Tregs (Table 4). Naive Tconvs significantly increased their mitochondrial parameters when cycling. There were no statistically significant differences in these measures between INRs, IRs, and HCs. Memory Tconvs also gained some mitochondrial mass when cycling, but cells from PLWH did so less effectively. However, all clinical groups exhibited a loss of Tconv mitochondrial transmembrane potential. This decrease was minimal in healthy controls, but more pronounced in PLWH, especially INRs, with a drop of up to 29.0%.

A hallmark of immunological non-response to ART is high CD4⁺ T-cell turnover, mainly involving memory cells [46, 47]. Similar trends are observed in various lymphopenic conditions [27, 48]. Earlier investigations in animal models and drug-induced lymphopenia showed that lymphocyte deficiency leads to activation and cycling of both conventional and regulatory T-cells [33, 49, 50, 51]. Our data confirm this relationship, reinforcing the concept of a homeostatic mechanism that helps maintain equilibrium between activated conventional T-cells and Tregs [40].

Insights from animal models, adoptive transfer studies, and human cell findings suggest that not only thymus-derived Tregs are involved in the homeostatic mechanism. Under lymphopenic conditions, memory CD4⁺CD25^– T-cells can rapidly proliferate and give rise to suppressive CD4⁺CD25⁺FOXP3⁺Tregs [52, 53]. These peripherally-derived Tregs have high division rates, shortened telomeres, and low telomerase activity, indicating their dependence on conventional memory T-cells and limited self-maintenance capacity.

Experiments in lymphopenic mice have shown that IL-2 may play an important role in balancing Tconv and Treg. IL-2 is crucial for expanding both T-cell subsets [34, 54]. While dividing conventional CD4⁺ T-cells secrete IL-2 for their own needs, the cytokine preferentially stimulates peripherally-derived Treg proliferation by binding to their high-affinity CD25 receptor [40, 54]. This feedback mechanism appears to stabilize the T-cell subset ratio, rather than maintaining their absolute numbers.

Human T-cells that express the transcription factor FOXP3 are heterogeneous. They can be divided into three subsets: (1) Naive Tregs formed in the thymus [55]. These cells have a CD45RA⁺CD25⁺⁺FOXP3^lo phenotype and express Helios transcription factor [56, 57]. (2) Mature Tregs differentiated from conventional CD4⁺ T-cells [53]. These cells have a CD45RA^–CD25^+⁣++FOXP3^hi phenotype [56]. (3) Mature non-suppressive cells in which FOXP3 expression is only transiently induced by stimulation [58]. These cells have a CD45RA^–CD25⁺⁺FOXP3^lo phenotype [56]. Upon stimulation, the suppressive Tregs (subsets 1 and 2) upregulate FOXP3 and proliferate actively. In contrast, the non-suppressive effector cells (subset 3) downregulate FOXP3 when they enter the cell cycle. This indicates that cells with Treg phenotype that upregulate FOXP3 upon cycling can be considered as bona fide Tregs.

Many researchers report high frequencies of Tregs in immunological non-responders [59, 60, 61, 62]. However, this study suggests that not all Treg subsets are equal. When CD4⁺ T-cells were divided into naive, central memory, effector memory, and TEMRA, then into quiescent and activated/cycling cells, increased Treg percentage in INRs was found to be driven by quiescent effector memory Tregs. This might be explained by prior findings that colon Tregs, mostly of effector memory phenotype [63], are less susceptible to Simian Immunodegiciency Virus (SIV)-infection than non-Tregs in SIV-infected macaques [24]. Noteworthy, the opposite is seen in activated/cycling CD4⁺ T-lymphocytes of immunological non-responders: Treg frequencies are deficient among naive and central memory cells. Naïve Treg deficiency might arise as a result of the decreased thymic output often found in INRs [64, 65]. In turn, central memory Tregs are primarily formed on the periphery, making it difficult to determine the cause of their deficiency. It is possible that CD4⁺ T-cells in INRs have functional issues that prevent the formation of peripherally-derived central memory Tregs. Thus, in immunological non-responders, there’s a Treg deficiency among activated/cycling naive and central memory T-cells, “masked” by increased Treg frequency in the quiescent effector memory compartment. This shift towards conventional CD4⁺ T-cells may weaken Treg control over Tconv cycling [66].

We found signs of Treg deficiency but couldn’t rule out Treg functional issues in immunological non-responders to ART. So, we checked the expression of molecules determining Treg suppressor activity: FOXP3 and CD25. Treg counts and function rely on signaling through CD25 [40, 67]. IL-2 signaling supports FOXP3 expression, Treg differentiation, expansion, and suppressor function [40, 68, 69]. Previous studies showed that in treatment-naive HIV-infection, CD25 expression on Tregs can be downregulated, disrupting IL-2-induced Treg expansion and shifting the balance between regulatory and conventional CD4⁺ T-cells [70]. Noteworthy, in our study, naïve and memory activated/cycling Tregs from both INRs and IRs showed equal or increased CD25 and FOXP3 expression compared to healthy controls. Thus, the phenotypic analysis didn’t indicate functional defects in Treg subsets of immunological non-responders to ART.

Noteworthy, no functional abnormalities were found when performing a standard Treg suppression assay. Tregs received from the INRs’ blood were able to effectively suppress the early activation of autologous effector CD4⁺ T-cells. In fact, the Tregs from INRs were even more suppressive than those received from IRs. These findings align with an earlier work by Shahbaz et al. [12], which noted that Tregs in certain cohorts of PLWH can upregulate pathways associated with a more suppressive phenotype. However, Karlsson et al. [71] revealed in animal models that the in vitro suppressive potential of peripheral Tregs correlates with preserved CD4⁺ T-cell counts and reduced T-cell activation, a pattern that doesn’t apply to INRs. Apparently, a deficiency of activated/cycling Tregs might play a key role in pathological CD4⁺ T-cell proliferation seen in INRs.

The function of Tregs depends on the fitness of their mitochondria [72, 73, 74]. These cells rely on fatty acid oxidation and oxidative phosphorylation to induce differentiation and sustain suppression capacity, stability, and survival [72, 75, 76]. Deregulation of mitochondrial function increases oxidative stress, induces autophagy, and mediates cellular damage and death. Previous research by our group found mitochondrial abnormalities in activated/cycling memory CD4⁺ T-cells in immunological non-responders to ART [26]. Specifically, we examined memory CD4⁺ T-cell transcription profiles and observed significantly reduced expression of genes involved in various stages of mitochondrial function: assembly of the respiratory chain, fatty acid oxidation, energy production, NAD⁺ generation, etc. It can be hypothesized that aberrations within the CD4⁺ T-cell population may be passed down to the Tregs derived from this subset. In the current analysis, we found no significant differences in mitochondrial mass or transmembrane potential between activated/cycling Tregs of PLWH and healthy controls. This is consistent with a prior study that reported no differences in mitochondrial mass and charge between antiretroviral-treated PLWH and healthy individuals in naive and memory CD4⁺ T-cells [77].

Mitochondria remodel during cell division, affecting cell proliferation and survival [44, 78, 79]. To assess mitochondrial functionality in immunological non-responders, we examined changes in mitochondrial mass and transmembrane potential in naive and memory Tregs transitioning from a quiescent to an activated/cycling state. This allowed us to evaluate mitochondrial adaptation to changing energy demands in healthy subjects and compare results with PLWH. We found that in INRs, the functional activity of mitochondria in memory Tregs is impaired, as evidenced by a decrease in transmembrane potential occurring independently of alterations in mitochondrial mass. This finding is important as the inhibition of the mitochondrial electron-transport chain was shown to inhibit proliferation and induced Treg formation, as found in mice [80].

Importantly, we found similarities in mitochondrial abnormalities of activated/cycling Tconvs and Tregs. This suggests the deep interdependence between these cell populations. It appears that Tregs, upon their formation from central memory cells, inherit all the defects present in their parent subset, which may impact both the quantity and functionality of newly formed lymphocytes. The mitochondrial impairment is critically important given that central memory cells represent the main reservoir for regeneration of effector CD4⁺ T-cell subsets during lymphopenia [81, 82]. Therefore, the mitochondrial defects we have identified in the activated/cycling memory Tconv and Treg cells likely have far-reaching consequences for the overall reconstitution of the CD4⁺ T-lymphocyte pool in immunological non-responders. Further investigation into the mechanisms underlying these mitochondrial alterations in Tconvs is warranted. And while Mitotracker dyes are a widely-used and valid approach to assess the mitochondrial parameters, direct measurement of mitochondrial function using the extracellular flux (Seahorse) assay may provide additional insights into the impact of mitochondria on Treg function and CD4⁺ T-cell homeostasis in the setting of persistent lymphopenia.

The study highlights the complex Treg dysregulation in immunological non-responders to ART. These patients have high Treg frequencies but lack activated/cycling Tregs in naive and central memory CD4⁺ T-cell subsets, indicating an imbalance that affects CD4⁺ T-cell recovery. Even with a normal phenotype, the activated/cycling memory Tregs in immunological non-responders show impaired mitochondrial function, as evidenced by decreased ability to tune the transmembrane potential in order to meet the energy demands, likely affecting Treg fitness and suppression. These Treg abnormalities may be inherited from the conventional parent population, as two subsets appear to be closely linked. Taken together, these findings emphasize the need to better understand Treg dynamics and function in CD4⁺ T-cell regeneration and suboptimal CD4⁺ T-cell recovery during HIV-infection.

ART, antiretroviral therapy; FMO, fluorescence minus one; HC, healthy control; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IL, interleukin; INR, immunological non-responder; IR, immunological responder; MFI, median fluorescence intensity; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cell; SIV, simian immunodeficiency virus; Tconv, conventional T-cell; TEMRA, effector memory cells re-expressing CD45RA; Treg, regulatory T-cell.

All data reported in this paper will be shared by the corresponding author upon request.

SY and KS designed the research study. ES, LK and SY performed the research. KS, ES and VV analyzed the data. KS, ES, LK, SY and VV wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The research protocol was approved by the Institutional Review Board of the Perm Krai Center for the Prevention and Control of AIDS and Infectious Diseases (IRB00008964). All participants provided written informed consent. The study was carried out in accordance with the guidelines of the Declaration of Helsinki.

This work was carried out within the framework of the State assignment ‘Investigating the functional activity of leukocytes and tumor cell lines during chronic infections and in response to chemical and biological compounds’ (registration number 124021900006-5).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/j.fbl2912429.