Linear regression analyses to assess the effect of age did not yield r2 values close to unity, with statistically non-significant p-values for the evaluated cellular populations (data not shown). These findings are consistent with the results reported by Villegas et al. regarding NKT cells and by Kokuina et al. for NK cells in two studies conducted on a Cuban adult population (15, 16).
There was not influence of sex on the values of NKT cells in older adults. Rojas-Pandales et al. also did not find a sex effect on NK and NKT values in Colombian individuals, although it is worth mentioning that they only studied subjects between 18 and 58 years of age (11). However, Villegas et al. found a sex effect on the NKT population, with a slightly higher number in males. On the other hand, a study by Apoil et al. showed a sex effect on NK cells in a French adult population, with significantly higher values in males, but no age effect was observed for this subpopulation (18).
Only 30% of the evaluated older adults were male, which results in an unbalanced representation of this sex, and the sample size was much smaller compared to the aforementioned studies. These factors may explain why these effects were not observed in the presented results. However, in the comparison of absolute values of NK cells in males, the p-value was close to 0.05 (p = 0.0697), and the mean was higher in both absolute and relative values, indicating that a larger sample size might reveal the sex effect.
Generally, women exhibit stronger humoral and cellular immune responses than men, linked to higher antibody levels and immune mediator production. Sex chromosome genes and sex hormones like estrogen, progesterone, and androgens influence immune responses, with estrogen enhancing both cell-mediated and humoral immunity, while androgens suppress Th1 cell differentiation and reduce IFN-γ production. Nutritional and microbiome differences also impact immune function between sexes (25–28). Taking into account that women participating in this study are over 60 years of age, the influence on the cellular immune response associated with the presence of estrogens would not be as much as significant.
Aging leads to decreased lymphocyte concentration and function. T lymphocyte decline is mainly due to thymic involution. Conversely, the increase of NK absolute and relative values in PB due to successive antigenic challenges is an evidence of the immunosenescence process. Subpopulation balance changes, with fewer naive lymphocytes and more memory, exhausted, and senescent phenotypes accumulate. Clonal diversity decreases, and oligoclonal populations expand, contributing to immunosenescence (4, 12–14, 22).
The values outside the range of normal values were found only above the upper limit of each reference value range for both NK cells and NKT cells. The high data dispersion observed in NKT cells, could be related to previous exposures to stimulating agents. The means of the absolute values for NK and NKT cells were higher in the group under 80 years old and in the male group. However, specifically for NKT cells, the standard deviation (SD) was higher than the mean in both distributions, indicating a high dispersion among the data. NKT cells accumulate in PB as their affinity antigens are encountered, so the interindividual variability found may be directly related to the number of previous exposures to agents capable of stimulating NKT cells (15). Additionally, the expansion of this population has been reported in pathological conditions such as sarcoidosis, allergies, various types of cancer, and HIV or Human Cytomegalovirus (HCMV) infection (15, 18). Apoil et al. found in the aforementioned study that HCMV seropositivity was associated with an increased frequency of CD56 + T cells (18). García et al. observed a high prevalence of HCMV infection in a group of Cuban adults aged 18 to 102 years (19). Considering this, it is possible that HCMV infection contributes to the increase in the NKT cell population in these groups.
The accumulation of terminally differentiated NK cells (CD56dim: with NKG2C+, CD57+, or FcεRIγ−) in PB can be caused by factors such as advanced age, chronic antigenic exposure following HCMV infection, or sex chromosome genes, as it has been found that males have higher values of these cell populations. Some sex differences in the maturation and differentiation process of NK cells persist in elderly females, where a higher NK CD56bright/CD56dim ratio has been reported compared to elderly males (17, 18, 20).
The 13.33% of the patients had malignant neoplasms, and within this percentage, there was one patient with relative and absolute values below the normal reference range, but only for NK cells. Among the most studied effector immune cells with biological significance in cancer are natural killer cells (NK cells) as part of innate immunity, further research into lymphocyte immunophenotypes has led to the discovery of new subpopulations with important roles in cancer biology, such as NKT cells. The effects of immunosenescence manifest rapidly in cancer and are associated with a worse prognosis, then with less immunosurveillance support, cancer incidence considerably rises with age (4, 6, 12–14).
The comparison according to neoplasia comorbidity showed that patients with neoplasia had considerably lower numbers of NK cells in PB compared to those with no neoplasia, while the NKT cells were reduced but not significantly. The OR indicates that adequate numbers of NK cells in PB might be a protective factor against malignant neoplasms. Arango et al. evaluated lymphocyte subpopulations in Cuban cancer patients and found that with increasing age, there is a tendency for a decrease in T, B, and NK lymphocyte populations, and patients with solid tumors showed the lowest values (17). Wang et al. demonstrate that age is one of the variables that most influences immunosenescence and they conclude that T cells, NK cells, and NKT cells are gradually affected in cancer (23).
NK cells play important roles in the antitumor immunity in the absence of sensitization, which contributed to the prevention of cancer invasion and metastasis and finally prevented the early dissemination of cancer cells (14). It is known that terminally differentiated NK cells increase in frequency in cancer patients, whereas NK CD56bright cells are in an exhausted state, which refers to the exhaustion or dysfunction caused by immunosuppression in the tumor microenvironment and tumor evasion mechanisms that result in a decreased ability of NK cells to effectively recognize and eliminate tumor cells. In this sense, NKT cells play an important supporting role, as they not only exert immunosurveillance against cancer but also help restore effector function and the recognition and elimination capacity of cancer cells by NK cells. NKT cells reverse NK cell exhaustion through cytokine production, expression of co-stimulatory molecules, and their immunoregulatory activity, reducing immunosuppression in the tumor microenvironment (17, 20).