COMPARISON OF DIFFERENT METHODS FOR EVALUATION CELLULAR IMMUNITY TO THE SARS-CoV-2 VIRUS

Most methods for evaluation T-cell immunity are laborious and unsuitable for routine laboratory diagnostics. This encourages researchers to create accessible and reproducible tests. The purpose of the study is to compare three methods for evaluation the level of cellular immune response to antigens of the SARS-CoV-2 virus in patients who have been ill and vaccinated against a new coronavirus infection. Examined: 26 people who had mild or moderate COVID-19 (group 1), 19 people vaccinated twice with Sputnik V, who did not have COVID-19 (group 2), 21 people who had COVID-19 and were twice vaccinated with Sputnik V (group 3) and 14 people who had COVID-19 twice (group 4). Peripheral blood mononuclear cells were isolated by gradient centrifugation. In the first method, mononuclear cells were incubated with the S-protein of the SARS-CoV-2 virus, stained with fluorescently labeled antibodies, the percentage of CD8highCD107a was counted on a BD FACS Canto II flow cytometer. When assessed by the ELISpot method on the “Human IFN-γ ELISpot” kit, IFN-γ production was stimulated by SARS-CoV-2 S-protein, or a mixture of SARS-CoV-2 protein peptides on the “Corona-T-test” kit. There were no significant differences in the level of expression of CD107a on CD8high in groups 1, 2, 3, and 4 and the number of IFN-γ producers per SARS-CoV-2 S-protein on the “Human IFN-γ ELISpot” kit. Production of IFN-γ is significantly lower in group 3 (hybrid immunity) 317.29±19.04 pg/ml than in groups 1 and 2 (post-infection and post-vaccination immunity) 454.95±20.32 and 470.77±26.24 pg /ml. The relative level of IFN-γ-producing cells in group 2 was higher (22.34±3.77) versus 16.83±2.35 in group 1 and 15.46±1.83 in group 3, the relative level of IFN- γ did not differ in these groups. Stimulation with full-length S-protein showed a significant reduction in the number of spots in group 4 (breakthrough immunity) 30.59±2.29 vs. 58.97±4.47 in group 3, and stimulation with a mixture of SARS-CoV-2 peptides in group 4 compared with group 3 revealed a significant increase in the number of IFN-γ-producing cells 86.72±7.20 versus 69.38±5.53 and IFN-γ production 991.25±65.18 pg/ml versus 760.76±50.70 pg/ml and in relative terms, 10.30±2.77 versus 8.61±2.66 and 68.10±9.41 versus 48.35±8.15, respectively. The results of three methods for evaluation the cellular immune response correlate positively with each other, but with different strengths

Diagnostic value of anti-GP2 antibodies determined in serum and coprofiltrates in children with inflammatory bowel disease

Inflammatory bowel diseases (IBD), such  as Crohn’s disease (CD) and  ulcerative colitis (UC), are characterized by chronically recurring inflammation of intestinal wall and are associated with a significant decrease in the  quality  of life. A spectrum of genetic  variants  associated with  Crohn’s disease  is described. Intestinal dysbiosis (DB)  may be the triggering factor of the disease. Glycoprotein 2 (GP2), the main protein of pancreatic zymogen  granules, is secreted  into the intestines with digestive enzymes.  Anti-GP2 antibodies were found in the serum of patients with CD.  The aim of the present  study was to investigate  the levels of anti-GP2 antibodies in serum  and feces of children with IBD  compared with the DB group.  Serums  and coprofiltrates from 110 children (64 boys and 46 girls) at the age of 12.3 (2.6-17.9) years were studied; 36 patients with CD, 30 patients with UC.  A comparison group consisted of 44 patients with DB. IgG and IgA antibodies against GP2 were tested with ELISA. Nonparametric statistics methods are applied, the results are presented as percentages and medians (Me (Q0.25-Q0.75)). The serum levels of anti-GP2 IgA antibodies were 9.97 (3.35-13.45) U/ml for the CD patients, 6.08 (2.71-14.26) U/ml for UC and 2. 94 (2.29-6.41) U/ml for DB. The levels of anti-GP2 IgG antibodies in serum were 6.16 (3.26-18.4) U/ml for CD, 5.26 (2.97-7.52) U/ml for UC, and for DB 5.23 (2.53-8.85) U/ml. The cut-off  threshold concentration for anti-GP2 IgG antibodies was 13.8 U/ml, with sensitivity of 63.2%, specificity 100%, and for IgA 5.63 U/ml, with sensitivity of 60.5% and specificity of 78.8%, thus being lower than the calculated cut-off  for adults (20 U/ml). The levels of anti-GP2 IgG in coprofiltrates in children of comparison group  were 1.99 (1.26-3.04) U/ml; in the  patients with CD, 23.5 (16.15-29.3) U/ml, and  in children with UC, 20.45 (13.63-25.5) units/ml (p < 0.001). The cut-off  value amounted 8.0 U/ml, with 100% sensitivity  and  100% specificity.  Concentrations of anti-GP2 IgA in coprofiltrates of patients with IBD  did not significantly  differ from DB patients. Moreover, the concentration of sIgA in the coprofiltrates of patients with IBD  was significantly  higher than  their level in DB group. The anti-GP2 IgA/sIgA  ratio was significantly lower in patients with CD (0.326 (0.23-0.512)), and UC (0.327 (0.205-0.435)), than in patients with DB (2.332 (1.575-3.523)) (p < 0.001);  the cut-off  level was 0.784, with a sensitivity of 97.7% and specificity  of 98.6%. It is discussed, whether fecal anti-GP2 IgA antibodies should  be considered as protective, supporting intestinal homeostasis, whereas anti-GP2 IgG antibodies are pathogenetically significant  for development of IBD.  Thus, using a non-invasive method for determining anti-GP2 antibodies in stool, when exceeding the cut-off for IgG, and reduction of IgA/sIgA ratio below the cut-off, one may differentiate IBD from DB with a similar symptoms at the onset of disease, with 100% sensitivity and 100% specificity

Mononuclear subsets and cytokine profile of venous and capillary blood in patients with psoriasis and healthy people

Psoriasis is considered an autoimmune disease with a predominantly cellular mechanism for the development of disorder. Studies in immune pathogenesis of psoriasis were performed either in animal model, which is not just similar to humans, or the data were obtained in patients by means of skin window method, which is traumatic, or by examining venous blood. However, it is difficult to discern parameters of the local immune response in venous blood samples. We have attempted to find an adequate method which would be convenient both for the patient and for the researcher, in order to assess local immune processes occurring in the skin affected by psoriasis. We examined 20 patients with a verified diagnosis of psoriasis, the average age was 44.3 years. The control group included 15 healthy adults, with average age of 46.6 years. Capillary blood was taken by fingerprick, whereas, in psoriatic patients, the samples were taken near the psoriatic lesion at a final volume of 400 μL in two microvettes. Venous blood (3 mL) was taken from the cubital vein into a vacuum tube with EDTA. Clinical analysis of venous and capillary blood was performed in automated hematological analyzer. Immunophenotyping was performed by four-color staining of whole capillary and venous blood followed by lysis of erythrocytes. Cytofluorometry was performed using techniques and reagents from BD Biosciences (USA). Plasma cytokines were determined by multiplex approach (MagPix, BioRad, USA). Upon clinical analysis of blood, the difference between capillary and venous blood was not found, either in healthy group, or among patients with psoriasis. In healthy people, the subsets of mononuclear cells, did not differ between venous and capillary blood. The samples of capillary and venous blood in the patients with psoriasis showed significantly increased levels of double-positive lymphocytes (CD45RA+/CD45R0+), B lymphocytes and NKT lymphocytes (both for relative and and absolute values). A significant increase in the percentage of naive T lymphocytes, activated helpers (Thact) and Treg, as well as B1 cells and Breg, and a significant decrease in B2 lymphocytes was registered in capillary blood of the patients with psoriasis. In venous blood samples from psoriatic patients, only a significant increase in Thact, Treg, and Breg was revealed. In the capillary blood of patients with psoriasis, we found a significant increase in the levels of non-classical M2 monocytes and inflammatory Minfl monocytes, and a decrease in classical M1 monocyte levels; in venous blood of psoriatic patients, only an increase in inflammatory Minfl monocytes was revealed. In capillary blood, all the studied cytokines in psoriasis patients significantly exceeded the levels of corresponding cytokines in healthy controls, except of IL-10. The levels of this cytokine did not differ from healthy group. In venous blood, the levels of most studied cytokines in the group of patients with psoriasis did not differ from the group of healthy ones. Approximately two-fold increase was revealed for IL-4, IL-21, IL-23 and TNF. First, the subsets of mononuclear cells and the cytokine profile of capillary and venous blood of healthy people did not differ significantly. Secondly, our proposed method for determining the subsets of mononuclear cells and capillary blood cytokines profile from the area of psoriatic lesions may be used to monitor local immunity in the patients with psoriasis. This approach is significantly less traumatic than the skin window method and more informative than the studies of venous blood

INDIСATORS OF THE LYMPHOCYTE SUBSETS AS EFFICICIENCY PREDICTORS OF THERAPY WITH INHIBITORS OF TNFα IN CHILDREN WITH INFLAMMATORY BOWEL DISEASE

(IBD), who were for the first time treated with TNFα blocker (infliximab). Our aim was to determine prognostic informative value of the immunological parameters in order to assess the treatment efficiency. A comprehensive research included seventy children with IBD from 12 to 18 years old in the course of specific treatment (49 children with CD, 21 children with UC).The comparison group consisted of fifty healthy children of similar age who were subjected to a similar detailed examination. The patients were divided into two groups, depending on their therapeutic response following 1 year of biological therapy: the first group showed a persistent positive effect of the drug, and the second group exhibited only unstable effects of the treatment. We determined the contents of major and small subpopulations of peripheral blood lymphocytes before the first administration of infliximab. Immunophenotyping was performed by multicolor flow cytometry (FC 500), using the CD45, CD3, CD4, CD8, CD19, CD16, CD56, HLA-DR, CD5, CD161, CD127, CD25, and CD294 markers.We have revealed that the content of B lymphocytes was significantly reduced in children with unstable effects of therapy. By contrast, the B lymphocyte levels in children with persistent positive therapeutic effect did not differ from the comparison group. Analysis of the composition of the B lymphocyte profile showed an imbalance in the B1-to-B2 cell ratio, with decreased of B1 cell counts in IBD patients against the comparison group. In addition, the patients with unstable therapeutic effect showed a significant decrease in B2 cell numbers compared with a group with persistent effect and comparison group. The numbers of NK cells in IBD patients were found to be reduced against the comparison group. Assessment of T lymphocytes subsets revealed a number of features in the patients with minimal therapeutic effects, i.e., an increased level of activated T helper cells (CD4+CD25+CD127high) and Th17 lymphocytes (CD3+CD4+CD161+), as compared to children with stable effect of treatment and to the comparison group. Moreover, in children with minimal effects of therapy, the levels of Tregs within T-helper cell subsets were significantly higher than in the comparison group. By means of ROC analysis, we have identified most informative parameters for the groups with minimal versus persistent therapeutic effect, and showed a good quality for a discrimination model involving relative amount of Th17 cells, activated T helper cells and B lymphocytes. The number of Тh17 lymphocytes (% CD3+CD4+ lymphocytes) allowed to predict the effect of therapy with a TNFα blocker with high probability. The present study enables us to propose cellular immunity testing, as a promising tool for monitoring clinical state of IBD patients

Evaluation of mediators of fibrosis and angiogenesis in the blood serum of premature infants with bronchopulmonary dysplasia

In premature birth and postpartum damage to the developing lung, the processes of the formation of pulmonary vessels and alveoli are disrupted, leading to bronchopulmonary dysplasia (BPD). BPD is a multifactorial disease and the pathogenesis of lung tissue damage is still not fully understood. Studies of angiogenesis biomarkers can be informative for assessing the development of BPD. In this study we examined the blood serum of 65 premature infants aged 6 to 180 days of life; gestational age at birth was 23-33 weeks, body weight 480-1840 g, APGAR score 5-6. All children in the early neonatal period had respiratory distress syndrome, then 46 children formed and 19 did not form bronchopulmonary dysplasia. The concentration of the factors of angiogenesis and fibrosis was determined in blood serum by ELISA. There were no differences in the levels of angiopoietins 1 and 2, vascular endothelial growth factor VEGF-D, transforming growth factor beta TGF-β, thrombospondin-1. We observed a tendency to increasing the level of VEGF-A, which is a key regulator of angiogenesis and lung maturation; we regard this tendency as a favorable sign of lung formation. We found tendencies to increase of the adhesion molecule of endothelial platelet cells PECAM-1, interleukin 8 and connective tissue growth factor CTGF. CTGF expression is enhanced by artificial lung ventilation and exposure to high oxygen concentrations. We consider an increase of CTGF in BPD to be an unfavorable change, since the binding of CTGF to VEGF inhibits VEGF-induced angiogenesis. In children with BPD, we found a decrease in the level of platelet derived growth factor PDGF-BB, the median concentration was 3180 pg/mL in BPD versus 4782 pg/mL without BPD (p = 0.024). PDGF is an important factor in tissue regeneration and plays an important role in the formation of blood vessels. We assume the decreasing of PDGF concentration in BPD can lead to a violation of the alveolarization necessary for the formation of the structure of healthy lungs. Studies of angiogenesis factors will help to better understand the pathogenesis of lung damage in BPD

Features of parameters of cellular immune depending on the activity of foci of demyelination in children with multiple sclerosis

MS is a common disease of the central nervous system that leads to disability and reduced quality of life. The debut of disease in 3-5% of patients occurs in childhood and has a less favorable course compared to adults. MS is caused by the activation of autoreactive T cells in the breakdown of peripheral tolerance, which is normally controlled by regulatory T cells (Tregs). It is promising to study expression of CD39 and CD73 in Treg and Th17 populations to assess their suppressive activity. Aim is to evaluate content of major and minor lymphocyte populations and expression of CD39 and CD73 in CD4+ lymphocyte population in children with MS. 111 children with MS were examined, 66 with contrast-negative lesions on MRI (Group 1), 45 with contrast-positive lesions (Group 2). The comparison group consisted of 46 healthy children (Group 3). Content of T, B, NK lymphocytes, Treg (CD4+CD25highCD127low), Thact (CD4+CD25highCD127high), Th17 cells (CD3+CD4+CD161+); expression of CD39 and CD73 in Treg, Th17 and Thact was performed by flow cytometry. An increase in content of T helpers, a decrease in NK cells in patients in group 2 was revealed. An increase in number of Thact and Th17 lymphocytes was obtained in patients of both groups with MS. Number of Tregs in group 1 was significantly higher than in group 3. Ratio of cells expressing CD39 and CD73 in MS patients depended on lymphocyte population as well as in the group 3. The highest content of CD39+ cells was observed in Treg population, and the lowest in Thact population. For CD73 expression, on the contrary, the highest expression of CD73 was observed in Thact cells, the lowest in Treg. When comparing groups of patients, it was found that in patients of group 1, number of cells expressing CD39 ectonucleotidase was significantly increased, and number of supTh17 was comparable with group 3. In both groups of MS patients, an increase in CD73 counts in Treg, Thact and Th17 was observed. Thus, informative populations of lymphocytes (CD4+ cells, Treg, CD39+Treg, supTh17) have been identified, which can be used to monitor condition of children with multiple sclerosis

Nuclear transcription factor kB (NF-kB) activity in lymphocyte populations in children with Wilson-Konovalov disease

Wilson's disease (WD) is a rare hereditary disease caused by a deficiency of the ATF7B transporter. The accumulation of copper can cause damage to organs and cells, mainly the liver. Copper exposure can modulate cytokine synthesis through molecular and cellular signaling pathways, including the nuclear transcription factor NF-kB pathway. NF-kB is the main regulator of inflammation and cell death, acts as a central link between liver damage, fibrosis and hepatocellular carcinoma. An excess of NF-kB-dependent cytokine response stimulates inflammatory reactions, but excessive inhibition of NF-kB can negatively affect the viability of hepatocytes. Method of flow cytometry with visualization — Amnis ImageStreamX allows to evaluate the activity of NF-kB (% of activated cells in cell populations). The aim: to evaluate the activity of NF-kB in lymphocyte populations in children with WD disease. Immunophenotyping of lymphocytes and assessment of the level of translocation of NF-kB were performed in 52 children with WD and in 25 children of comparison group. The mass concentration of copper in daily urine was determined by atomic absorption method using the AAnalyst 800 spectrometer. In children with WD, the content of cells with NF-kB translocation varied from 5 to 90% depending on the lymphocyte population; the highest level was detected in B cells — 57.5 (37-68) %. A significant difference in distributions of the number of cells with NF-kB translocation between WD and healthy children was shown (F-criterion, p < 0.01). In most cases, children with WD are characterized by a decrease in the activity of NF-kB in populations of B cells (in 43% of cases), T helper cells (48%), T cytotoxic (44%) and Th17 lymphocytes (41%). In children with WD, the concentration of copper varied from 9.7 to 2582 mcg/day, Me = 616 (210-1173). A direct relationship was obtained between the copper content in urine and the level of translocation of NF-kB in B lymphocytes, r = 0.34, p = 0.016. The activity of the NF-kB correlates with biochemical markers of the severity of liver damage (ALT, AST, GGT) and with copper content in urine. The study of the NF-kB signaling pathway seems promising for a better understanding of the pathogenetic mechanisms of the formation of inflammation and liver fibrosis in children with WD

Результаты трехлетней вакцинации детей против пневмококковой инфекции в России

Background. After inclusion of pneumococcal vaccination in the National Vaccination Schedule, it is very important to evaluate the efficacy of routine immunisation of the child population for more than 3 years. The obtained results provide opportunity to analyse the problems in achieving the goal, determine their causes, and suggest the ways of overcoming. Our aim was to study the results of a three-year period of pneumococcal vaccination of children. Methods. The quality of immunoprophylaxis of pneumococcal infection in the territory of the Russian Federation were assessed by analysing the coverage of vaccination and timeliness of its conduct after the inclusion of pneumococcal vaccine in the National Vaccination Schedule. The actual epidemiological efficacy of pneumococcal vaccination was assessed based on morbidity and mortality due to community-acquired pneumonia, incidence of acute otitis media among children. By questioning parents (n = 352) who applied to the Federal State Autonomous Institution of the Russian Federation Ministry of Health ‘National Medical Research Centre for Children’s Health, the timeliness of pneumococcal vaccination for infants was established. Results. In most regions, a high level of pneumococcal vaccination coverage was reached (87% of children). Despite the fact that the majority of children (73%) were vaccinated untimely. In particular, the results of a questionnaire survey conducted in the Moscow vaccination centre indicate insufficient awareness of parents for the need to vaccinate infants against pneumococcal infection by primary care professionals and, as a consequence, a low level of timely initiated vaccine introduction (40.1%). The introduction of routine prophylactic pneumococcal vaccination in Russia resulted in a 35% reduction in the death rate of children from community-acquired pneumonia, led to a decrease in the incidence of acute otitis media. Conclusion. The introduction of routine prophylactic vaccination of children against Streptococcus pneumoniae helps to reduce morbidity and mortality from pneumococcal infections. The surveillance system for community-acquired pneumonia requires further improvement. It is advisable to conduct an additional analysis on the reasons for refusals and medical exemptions to vaccination. It is important to increase the professional level of paediatricians in prophylactic vaccination.Обоснование. После включения в Национальный календарь профилактических прививок вакцинации против пневмококковой инфекции очень важно оценить эффективность проводимой более 3 лет рутинной иммунизации детского населения. Полученные результаты позволят проанализировать проблемы в достижении цели, установить их причины и предложить пути преодоления. Цель исследования — изучить результаты трехлетнего периода вакцинации детей против пневмококковой инфекции. Методы. Проведена оценка качества иммунопрофилактики пневмококковой инфекции на территории Российской Федерации путем анализа охвата прививками, своевременности их проведения после включения пневмококковой вакцины в Национальный календарь профилактических прививок. Выполнена оценка фактической эпидемиологической эффективности вакцинации против пневмококковой инфекции на основании заболеваемости и смертности внебольничными пневмониями, заболеваемости острым средним отитом среди детского населения. Путем анкетирования родителей (n=352), обратившихся в ФГАУ «НМИЦ здоровья детей» Минздрава России, установлена своевременность вакцинации младенцев против пневмококковой инфекции. Результаты. В большинстве регионов достигнут высокий уровень охвата детей прививкой против пневмококковой инфекции (87%). При этом большинство детей (73%) были вакцинированы несвоевременно. В частности, результаты анкетирования, проведенного в центре вакцинации г. Москвы, указывают на недостаточную информированность родителей о необходимости вакцинации младенцев против пневмококковой инфекции специалистами первичного звена и, как следствие, низкий уровень своевременного начала введения вакцины (40,1%). Внедрение плановой вакцинопрофилактики против пневмококковой инфекции в России позволило на 35% снизить смертность детей от внебольничных пневмоний, привело к уменьшению заболеваемости острыми средними отитами. Заключение. Внедрение плановой вакцинопрофилактики детей против Streptococcus pneumoniae способствует снижению заболеваемости и смертности от пневмококковых инфекций. Система эпиднадзора за внебольничными пневмониями требует дальнейшего совершенствования. Целесообразно проведение дополнительного анализа причин отказов и медицинских отводов от вакцинации; важное значение имеет повышение профессионального уровня врачей-педиатров по вопросам вакцинопрофилактики.КОНФЛИКТ ИНТЕРЕСОВЛ.С. Намазова-Баранова — получение исследовательских грантов от фармацевтических компаний Пьер Фабр, Genzyme Europe B. V., ООО «Астра зенека Фармасьютикалз», Gilead / PRA «Фармасьютикал Рисерч Ассошиэйтс СиАйЭс», Teva Branded Pharma ceuti cal products R&D, Inc / ООО «ППД Девелопмент (Смоленск)», «Сталлержен С. А.» / «Квинтайлс ГезмбХ» (Австрия).М.В. Федосеенко — получение гонораров от компаний Pfizer, Sanofi Pasteur, MSD за чтение лекций.Остальные авторы статьи подтвердили отсутствие конфликта интересов, о котором необходимо сообщить

AGE-DEPENDENT FEATURES OF EVOLVING HUMORAL IMMUNITY IN CHILDREN

Abstract. Age dynamics of humoral immunity was studied in healthy children, i.e., 11 newborns, 33 infants of 4 to 8 months, 32 children of 1 to 2 years old,, 17 children of 4 to 5 years old, 25 children of 6 to 8 years old, 15 children of 9 to 11 years old, and 28 adolescents of 14 to 16 years old. Evaluation of membrane receptors on B cells was performed by means of three-colour fluorescent label and allowed of characterizing B1 subpopulations (CD19+CD5+CD27-), naпve B2 cells (CD19+CD5-CD27-), and B2 memory cells (CD19+CD5-CD27+). B1 cells have been shown to dominate in blood of newborns and younger children (up to 5 years old). By the contrary, B2 memory cells were nearly undetectable in newborns, and exceeded 20% in adolescents (by 15 years old). Meanwhile, it has been revealed that the amounts of IgG1 and IgG3 subclasses did progressively increase with age, whereas IgG2 remained decreased to 50% of adult values for a long time, and reached them by 11 years and later. We suggest that the age dynamics of IgG subclasses is connected with age-dependent changes in B cell subpopulations

AGE-DEPENDENT CHANGES OF T-REGULATORY AND Th17 SUBSET LEVELS IN PERIPHERAL BLOOD FROM HEALTHY HUMANS

Age-dependent development of Th17 and Treg lymphocyte subsets in healthy  humans is studied insufficiently. The  present  study aimed  to investigate  quantitative characteristics of Th17  and  Treg subsets in peripheral blood of healthy  subjects for various age groups.352 healthy  humans (168 female  and 184 male), one month to 85 years old, were subject to examination, including 79 infants in their first year of life; 34 children at aged 1 year to 2 years 11 months; 24, at 3 to 4 years 11 months; 28, at the age of 5 to 6 years 11 months; 25 children aged 7-8 years 11 month; 36 children aged 9 to 11 years 11 months; 39 adolescents aged 12 to 14 years 11 months; 26 adolescents aged 15 to 18 years; 25 young adults aged 20 to 35 years; 11 adults at 36 to 49 years old; 16 adults aged 59-70 years, and 9 elderly people over 70 years old. The study was performed with capillary blood in children under 2 years, and venous blood taken in elder persons.  The basic and ‘minor’ subsets of peripheral blood lymphocytes were evaluated by flow cytometry using four-color staining of whole blood and following erythrocyte lysis. We used the following surface markers: CD3, CD4, CD8, CD25, CD127, CD161, CD45R0 for lymphocyte subsets detection.It has been  shown  that  Treg percentage (a ratio  of CD4+CD25hiCD127low/neg  in the CD3+CD4+  gate) did not  depend on  age of the  people  under  study, and  can  be approximated by a linear  function. The  absolute number of Tregs in childhood is progressively  decreased and,  after 10 years old, it reaches  plateau  values. This age-dependent relationship may be approximated by a logarithmic function. Evaluation of Th17 subset levels demonstrated a strong  relative  and  absolute  age-dependent growth  of this  cell  subpopulation. Percentage and absolute  numbers of Th17 lymphocyte (share  of CD4+CD161+CD45R0+ in the CD3+CD4+gate), can be approximated by a square function. The age of 10-12 years seems to be critical to the immune system formation. We suppose  the process of the immune system development to be completed at this age, and maturation of the immune cell populations is then  observed.  A decrease in both relative and absolute  numbers of Treg and Th17 lymphocyte subsets was found in elderly persons (> 70 years old). Our data on peripheral blood Tregs and Th17 subsets, with respect  to their percentage and absolute  numbers in healthy  humans, may be used as age-related reference values