Participants. Ninety-nine participants were recruited from the University of the Third Age (U3A), which is an organization encouraging the elderly over 55 years of age to stay active by participating in many educational programmes, including arts, classical studies, discussion classes, computer courses, crafts, drama, film/cinema studies, history, languages, literature, music, sciences, social sciences, and physical activity. The current health status and lifestyle of the participants were controlled by using the health history questionnaire . The age of 60-90 years and the same access to medical healthcare were the inclusion criteria. The exclusion criteria, based on the medical interview, were as follows: acute infectious and oncologic diseases, cardiovascular, neurological and musculoskeletal disturbances and an implemented pacemaker. Moreover, twenty-two older adults withdrew from the project during the study due to high blood pressure above 180/110 mmHg, hospitalization, serious knee injury or a cold. Eventually, the study included fifty-four individuals aged 65-88 years (females n=47, males n=7) who represented the successful ageing according to the definition by Geard et al. . According to the gait speed measurement (6-min walk test), the Åstrand-Ryhming bike test and Community Healthy Activities Model Program for Seniors (CHAMPS) questionnaire, the participants were classified into physically active (n=34) and inactive (n=20) groups (Table 1). All participants were informed of the aim of the study and gave their written consent for participation in the project. The protocol of the study was approved by The Bioethics Commission at Regional Medical Chamber Zielona Gora, Poland (No01/66/2017, No21/103/2018) in accordance with the Helsinki Declaration.
Table 1. Anthropometrics and body composition as well as physical activity level (mean ± SD).
Body composition. Body mass (BM) and body composition fat-free mass (FFM) and fat mass (FM) were estimated by a bioelectrical impedance method using Tanita Body Composition Analyser MC-980 (Japan) calibrated prior to each test session in accordance with the manufacturer’s guidelines. Duplicate measures were taken with the participants in a standing position; the average value was used for the final analysis. The recurrence of measurement was 98%. The measurements were taken between 7:00 and 9:00 a.m., before blood sampling.
Functional fitness. The 6-min walking test (6MWT) was performed according to technical standards of European Respiratory Society and American Thoracic Society . A marked walkway was laid out in a 50-m rectangular area (dimensions: 20 x 5 m), with cones placed at regular intervals to indicate the distance covered. The aim of the test was to walk as quickly and as far as possible over a span of six minutes. The participants were allowed to self-pace (a preliminary trial was useful to practice pacing) and rest as needed. The total distance walked in the test was recorded and the 6MWT gait speed was then calculated by the following equation: 6MWT gait speed (m/s) = total distance(m)/360s. The gait speed ranging from 1.3 to 1.8 m/s classified the participants as physically active and the gait speed <1.3 m/s classified them as inactive according to Middleton et al. .
Cardiorespiratory fitness. The measurement of maximal oxygen consumption (VO2max) was performed via the indirect method known as the Åstrand-Ryhming bike test (6-min submaximal exercise test) which relies on the linear relationship between heart rate (HR) and VO2 to predict maxVO2 and which is recommended for both men and women of various ages . Each participant performed a 6-min submaximal exercise test on a cycle ergometer eBike GE Healthcare (Germany). Initially, the study participants rested for 15 minutes prior to the measurement of their resting HR. The seat height and handlebars were adjusted for each participant prior to the test. According to normative data for submaximal exercise test, the participants who reached the values of VO2max>35 mL/kg/min were classified as active (high activity level) and the remaining ones were determined as inactive (average and low activity level).
Type and amount of physical activity. The type and weekly amount of physical activity was evaluated by CHAMPS questionnaire . The CHAMPS was originally designed to assess the types and intensity levels of physical activity including lighter (e.g. leisurely walking, water gymnastics, stretching, Tai-Chi) as well as more vigorous activities (e.g. dancing, cycling, swimming). Currently, the CHAMPS also includes a group of items related to a sedentary lifestyle e.g. sitting and chatting with friends.
Blood sampling. Blood samples were taken from the median cubital vein using S-Monovette-EDTA K2 tubes (Sarstedt, Austria) for flow cytometry analysis and morphology and S-Monovette - serum tubes were used for other biochemical markers. Serum samples were left to clot for 45 min before centrifugation and then centrifuged at 3000 g and +8oC for 10 min. Aliquots of serum were stored at -80°C.
Flow cytometry analysis. Cytometric analysis was performed using eight-parameter CyFlow Space Sorter flow cytometer by Sysmex Partec (Germany). For the analysis of immune cells, CyLyse kit by Sysmex (Germany) was used. 100 µl venous blood was mixed with fluorochrome labelled monoclonal antibodies (CD8 APC, CD4 FITC, CD45 RA Pacific Blue™ CD45RO PE) and incubated for 15 minutes in the dark at room temperature. After the incubation 100 µL of Leukocyte Fixation Reagent A was added and incubated again in the dark for 10 minutes. In the last step, 2.5 ml Erythrocytes Lysing Reagent B was added, mixed and incubated in the dark for 20 minutes and further measurements were made. T helper and cytotoxic lymphocytes were gated by positive surface staining for CD4 and CD8 and were expressed as a percentage of gated lymphocytes. Memory and naïve subpopulations were gated by positive surface staining for CD45RO and CD45RA, respectively. The strategy of gated lymphocytes T was shown in Figure 1. The ratios of CD4+CD45RA+ to CD4+CD45RO + and CD8+CD45RA+ to CD8+CD45RO+ as prognostic markers of chronic diseases, were calculated according to Hang et al. . The CD4/CD8 ratio was calculated according to McBride and Striker  and the reference values were adopted from Strindhall et al. . The ratios ≥1 or ≤2.5 are generally considered normal, however, a wide heterogeneity exists because of sex, age, ethnicity, genetics, environmental exposures and infections. The inverted or high CD4/CD8 ratio (<1 or >2.5) is regarded as an immune risk phenotype and is associated with immunosenescence and chronic inflammatory diseases .
Figure 1.Gating strategy to identify the CD4+ and CD8+ T lymphocyte and the frequency of CD4+ and CD8+naïve and memory T lymphocytes.
Haematological variables. Peripheral blood morphology: white blood cell count (WBC), granulocytes (%GRA), lymphocytes (%LYM), mid absolute count (%MID), red blood cells count (RBC), haemoglobin (HB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets (PLT) were determined by using 3 diff BM HEM3 Biomaxima (Poland).
Biochemical markers. Total cholesterol (TC), high-density lipoproteins (HDL), low-density lipoproteins (LDL), triglycerides (TG) were determined by using BM200 Biomaxima (Poland). The non-HDL cholesterol was calculated by subtracting HDL from the total cholesterol concentration. The CRP was measured using a high sensitivity assay in duplicate by means of commercial kit from DRG International (USA) with the detection limit of 0.001 mg/L. The intra-assay coefficient of variation (intra-assay CV) for the CRP ELISE kit was 4.44%, and the inter-assay coefficient of variation (inter-assay CV) was 3.28%. The serum glucose was evaluated by using commercially available reagents and mobile spectrophotometer DP 310 Vario II (Germany).
Cytomegalovirus (CMV) IgG. The CMV IgG serostatus was determined using commercial kit from DRG International (USA). The reference values of CMV-seronegativity (IgG CMV-) were <9 DU/mL and CMV-seropositivity (IgG CMV+) >11 DU/mL. The intra-assay CV for the CMV kit was 7.75% and inter-assay CV was 11.45%.
Statistical analysis. Statistical analyses were performed using the R system, version 3.6.1 . The assumptions for the use of parametric or non-parametric tests were checked using the Shapiro-Wilk and Levene’s tests to evaluate the normality of the distributions and the homogeneity of variances, respectively. The significant differences in mean values between the groups (active vs. inactive) were assessed by the one-way ANOVA. The analysis of covariance (ANCOVA) was used to demonstrate the body composition and functional fitness variables that might influence the covariate variables such as CMV IgG concentration and T lymphocyte phenotypes. The chi-squared (c2) test was used to compare females and males using categorical data from CHAMPS questionnaire. If the normality and homogeneity assumptions were violated, the Mann-Whitney non-parametric test was used. Additionally, eta-squared (η2) was used as a measure of effect size which is indicated as having no effect if 0 ≤ η2< 0.01, a minimum effect if 0.01 ≤ η2< 0.06, a moderate effect if 0.06 ≤ η2< 0.14, and a strong effect if η2 ³ 0.14 [30,31]. Spearman’s rank correlation (rs - Spearman rank correlation coefficient) was calculated to describe the relationships between body composition and T lymphocyte count. Statistical significance was set at p<0.05.