Since the immune function is a marker of health and longevity and a positive relation has been shown between a good function of several immune cells and health status, we considered several immune function parameters associated with gender and body composition to be adequate biomarkers of the biological age. As mentioned in the introduction, changes in the elderly regarding acquired immunity associated with the T cells pool mainly involve a reduction in the number of naïve T cells, an increase in the number of memory T cells [15,16] and a diminished response to vaccines . One of the major findings of the current research is that ageing-related adaptation of the immune system seems to result from a reduced repertoire of naïve CD4+ and CD8+ T cells due to the thyme involution as well as from prolonged exposure to different antigens throughout life and the accumulation of memory CD4+ and CD8+ which compensate for a number of naïve T cells. In our research a statistically significantly larger population of CD4+ helper lymphocytes was found in the elderly than in the younger subjects contrary to Alam et al. , who showed a high level of CD4+ in the elderly subjects. It is not entirely clear how age influences changes in the CD8+ T cells population. The present study found a tendency towards the presence of high percentage of CD8+ in the elderly but this difference did not reach the significance level. Nevertheless, Hirokawa et al.  observed a decrease in the number of CD8+ T cells with age. Changes in the T cell system affect the ability to mediate effective immune responses against new antigens and the ability has been proven to be reduced in the elderly in comparison to the young. An increase in the number of senescent T cells and a decrease in the number of naïve cells diminish the protection range and capacity against pathogens. Therefore, the issue of ageing populations prompts researches worldwide to keep trying for years to design an immune risk profile that determines a potential rate of ageing of the immune system, and thus the whole body. The assessment of the immune risks profile including analyses of changes in the number of specific subpopulation of immune cells may be relevant when predicting the occurrence of neoplastic, autoimmune and neurodegenerative diseases as well as metabolic disorders, such as type 2 diabetes, or cardiovascular diseases that are more common in the elderly .
The immunological risk profile, which, among others, included the analysis of prevalence of inverted CD4/CD8 ratio, was compiled with the participation of Swedes, Dutch and Belgians. Some of the observations are comparable, there are, however, some differences due to the lifestyle and environmental factors. In the OCTO/NONA project, the inverted CD4/CD8 ratio was found to be associated with increased mortality and a higher risk of infection. The BELFRAIL project in women reported the accumulation of CD4+naive T cells and the CD4/CD8 ratio > 5 to be negatively correlated with increased survival as opposed to CD4/CD8 ratio < 1, which showed a positive correlation with 3-year survival . The CD4/CD8 ratio in healthy individuals is poorly defined but a value between 1.0-1.5 to 2.5 is used as the norm. Research shows that many factors like: age, ethnicity, genetics, gender, exposures and infections may all impact the ratio. The inverted ratio may result from isolated apoptotic or targeted cell death of circulating CD4 cells, expansion of CD8 cells, or a combination of both. Some of the researchers cl
aim the incidence of inverted CD4/CD8 ratio increases with age. An inverted ratio has already been recorded in 8% of individuals aged 20–59 and in 16% of individuals aged 60–94 . In older adults with confirmed inverted CD4/CD8 ratio remodelling enumerative and functional immune changes have been observed and these included: reduced number of B cells (CD19+) and an expansion of late-differentiated or senescent T cells (CD8+CD28−), which are important T cells against cytomegalovirus (CMV). Some research suggests that inverted CD4/CD8 is an immune risk factor which could be associated with various pathologies and it is frequently associated with premature immunosenescence and repeated viral infections .
On the other hand, researchers like Strindhall et al.  showed that in surviving OCTO/NONA participants older than 100 years, no inverted CD4/CD8 ratio had been recorded from the age of 85 on and the mean CD4/CD8 ratios were found to increase with age. The increase of CD4/CD8 with age was also confirmed in the Japanese population . Interestingly, in Polish population we showed that in elderly individuals the inverted CD4/CD8 ratio occurred only in 60–74 age group and in the group of 75-90-year-olds the CD4/CD8 ratio ≥ 2.5 was identified in 67% of the individuals. In turn, in the Leiden 85 + study, only 2% of individuals aged 89 years who participated in the study showed an CD4/CD8 ratio < 1 compared with 20% in those between 70 and 81 years of age. The observed increase in the CD4/CD8 ratio with age was similar to the
results obtained by Vasson et al.  in the Austrian elderly but opposed to the results from Spanish or French population where a trend towards a decreasing CD4/CD8 ratio was observed. The differences between countries/regions can be related to lifestyle factors such as habits, nutrition, physical and mental activity.
The finding of an increasing prevalence of the CD4/CD8 ratio and the increased or decreased mortality should be confirmed in additional populations in other countries. The nature and cause of this immunosuppressed status is certainly an important topic for future research, but there is no doubt that whether the CD4/CD8 ratio in older people decreases or increases with age also depends on gender. Previous studies by Wikby et al.  and Hirokawa et al.  reported that the inverted CD4/CD8 ratio concerned mainly men, while the number of naïve cells CD8 + and the CD4/CD8 ratio were significantly higher in women. We also observed a significantly higher percentage of naïve CD4+ and CD8+ T cells in women compared to men. The inverted CD4/CD8 ratio was detected in 5 out of 83 examined women while the ratio ≤ 1 was found in 4 out of 16 examined men. This is not only the consequence of the thymus atrophy but also hormonal changes. The effect of the hormones on the CD4/CD8 ratio was confirmed by the correlation between low plasma estradiol levels, high CD8+ values and low CD4/CD8 ratio . In our study, the majority of the study subjects (84%) were females with only 16% being males and yet, approx. 25% of the male group showed CD4/CD8 ratio < 1. To provide more substantial evidence, however, the next stage of the study should include a larger male sample. An impaired immune status may increase a risk of cardiovascular diseases including hypertension. The prevalence of hypertension in our subjects was similar in women and men and it amounted to approx. 56–58%. A large body of evidence has suggested that innate and adaptive immune responses are involved in hypertension-mediated low-grade inflammation. The recent study on mouse models has revealed that lymphocytes may be involved in the regulation of T lymphocyte proliferation and the production of pro-inflammatory cytokines, which contribute to the hypertensive inflammatory response . In humans with hypertension, T lymphocytes polarise towards Th1-type CD4+T helper cells, which initiates inflammatory response to neoantigens resulting from hypertensive factors activity and the activation of the immune system . When determining the T cells phenotype in newly diagnosed hypertensive patients, Youn et al.  showed a significantly higher number of circulating immunosenescent pro-inflammatory CD8+ T cells in individuals with hypertension compared to healthy people. Itani et al.  showed higher values of circulating CD4+ CD8+ memory T cells in individuals with elevated blood pressure. In our study, we also observed higher numbers of the CD4 + and CD4+ memory cells in individuals with hypertension compared to the others.
According to Alonso-Fernandez and De la Fuente , the CD4/CD8 ratio could be useful as a marker of immune function in the elderly and also as a predictor of health status. In our study, a CD4/CD8 ratio > 2.5 was observed in approx. 52% of the subjects with hypertension. The other individuals suffered from additional diseases such as rheumatoid arthritis, diabetes, thyroid disorders. Therefore, it is difficult to determine whether the high values of memory CD4+ cells in this group were exclusively the effect of hypertension or the consequence of other disease entities interference. The explanation of this effect requires further investigation.
Adipose tissue lies at the crossroads of nutrition, physical exercise and immunity. An increase of fat content is accompanied by dynamic changes in immune cell populations. Nearly all immune cell studied to date are quantitatively altered by obesity with most immune cells increasing in adipose tissue. Obesity-induced changes in their number and activity result in the activation of local and later systemic inflammatory response, marking the transition from simple adiposity to diseases such as type 2 diabetes mellitus, arterial hypertension and ischemic heart disease. In our study, 50% of investigated seniors were classified as overweight and 29% as obese and FMI was found to be correlated with the numbers of CD4+ and CD4+CD45RO+. Both CD4+ and CD8+ T cells have been shown to increase with obesity and preceded macrophage infiltration [30,31]. Further evidence demonstrated that it is the T-cells rather than the macrophage that initiate adipose tissue inflammation and dysfunction .
There are some limitations of the present study with the first one being a relatively small sample size, especially in the male group, which made it difficult to reach definitive conclusions. Secondly, the study is limited by a lack of information on the lifestyle and environmental factors, nutrition, physical activity and exposure to pathogens during lifetime. Therefore, in order to address these limitations, the study will be designed to increase the sample size and to explain the effect of adequate nutrition (dietary inflammatory index) and appropriate mental and physical activity as well as sleep quality.