RSV infection causes severe bronchiolitis in children and severe pulmonary disease in elderly individuals [1–5]. In this study we tried to understand the immunological mechanisms which could make elderly more susceptible to RSV reinfection and at risk of serious respiratory disease. Although after primary infection children develop a CTL response that clears the virus and should protect against reinfection for many years, it is still not clear why this virus is associated with a high reinfection rate and high morbidity in the frail elderly or in people with cardiopulmonary disease [3–6].
The progressive age related decline in immune memory has been proposed as a fundamental mechanism for this phenomenon. It is essential that the RSV specific memory T cells persist after primary infection to keep enduring immunological protection. The mechanisms behind the development of long-lasting memory cells are incompletely understood but, recently, it was shown that IL-7Rα+ effector cells are able to survive antigen deprivation and develop into long living memory CD8+ T cells [22, 36]. To this aim, we characterized the CD8+ CD127+ long-lasting memory cells specific to RSV by pentamer in healthy subjects of three different age groups. This analysis was performed by evaluating the immunological parameters in human PBMCs at baseline and after in vitro stimulation with a specific RSV peptide to partially mimic the natural events occurring after in vivo reinfection.
However, it is worthy to note that no cytokines were added to the stimulated lymphocytes in order not to interfere with the microenvironment and cell proliferation. Therefore, although environment factors such as IL-7 or IL-15 may be fundamental to expand memory cells , these were not considered in this study to rule out possible artefacts and to exclude the risk of using the same amounts of cytokines for all the subjects without considering the variability due to age.
In the present study, we analysed the different CD8+ memory cell populations and focused on the long-lasting memory subset in the presence or absence of RSV antigen. For PBMCs stimulation, we used an RSV peptide derived from the nucleocapsid antigen, able to stimulate human HLA-A(*)02.01+ peripheral blood mononuclear cells, as previously shown . It is worthy to note, however, that a number of virus specific T cells may be missed because they do not recognize the epitopes contained in the pentamer. On the basis of classical characterization of TCM, TEM, and TEMRA, we did not find differences among the three age groups, nor were differences based on age found for specific CD8+/CD127- T cells labelled with RSV pentamer. On the contrary, a clear decline of RSV specific CD8+/CD127+ cells was evident after antigen stimulation in the oldest group, indicating that this specific memory population tends to diminish with age and reaches very low levels when the subject is newly exposed to the virus. Moreover, a proliferative induction of this population is triggered in PBMCs of the young after Ag stimulation, while no induction is evident in the oldest group. This data indicates that RSV specific memory cells are able to re-expand following the antigenic boosting, but this capability decreases in size with increasing age. This was probably due to a down-regulation of T cell receptor (TCR) after some reinfections or because IL-7 level, which is fundamental for rejuvenating T cell immunity and improving the survival of memory T cells, declines with age . Moreover, Bcl-2, which is an anti-apoptotic molecule that is critically involved in the IL-7 mediated cell survival, appears to be less expressed, in the elderly, in RSV specific CD8+CD127+ memory cells which then differentiate in effector cells, indicating that a Bcl-2 down-regulation occurs with the age. Although other studies have shown that influenza virus and RSV specific cells retained a high IL-7Rα expression in the lung , hypothesizing that these cells were activated by their cognate antigen and might rely on IL-7, this data does not consider the age of the patients and does not explain whether these specific cells are recruited in the lung from the circulation. Therefore, trying to understand this evident decrease of RSV specific memory CD8+ T cells in the elderly, we analysed the frequency of CD4+CD25+FoxP3+ T cells and defined natural regulatory (Treg) cells which have a negative regulatory effect on immune responses [38, 39, 44, 45]. CD8+ T-cell effector functions and proliferation are often dampened by negative regulation by Treg cells which play a role in regulation of adaptive immune response to several viruses . In this study, the increase of Treg cells in the elderly, matched with a parallel increase of IL-10, that is an important suppressive cytokine, produced by a large number of immune cells in addition to the antigen-driven IL-10-producing regulatory and the naturally occurring suppressor CD4+ T cells . In fact, the amount of IL-10 expressed by PBMCs without Ag stimulation, in people > 60 years, was very high (252,2 ± 12,6 pg/ml) in comparison with the level of the youngest subjects (Table 4). The inhibitory effect of Tregs on Th1 response  was also demonstrated by a diminished IFN-γ production in older subjects in comparison with the younger and by a shift from Th1 cytokines (including IFN-γ and TNF-α) to Th2 cytokines (including IL-10) with aging. In this study, we observed that the Th1 response was also characterized by a marked decrease of the proinflammatory cytokine, TNF-α, in the elderly, and that this tended to further diminish after Ag stimulation. Consequently, it is likely that CTL response and the effector activity mediated by CD8+ T cells are no more efficient to combat the virus. Although recent studies have shown evidence that RSV immune response is mainly compartmentalized in the lung , the data presented here confirm a decrease of specific CD8+ memory cells with the age in the peripheral blood of healthy subjects. This could be due, in particular, to the high frequency of Tregs in old people that could partly suppress the specific immune response and to a weakness of the Th1 response, determining a greater frequency of RSV reinfection during the elderly. Moreover, since infection can and does occur in the presence of circulating antibodies, a balanced immune response including RSV specific neutralising antibodies would be necessary for maintaining protective immunity in the host. These results show, in addition, that there is a decline of RSV specific protective humoral response in elderly which, as in this case, could be partly due to the increase of Tregs. In fact, these cells are able to suppress the proliferation not only of CD8+ but also CD4+ lymphocytes , that interacting with B cells specific for peptide antigens can generate a T cell dependent antibody response. In this context, we believe that the antigen hierarchy may be completely subverted with a potential deletion of viral epitopes specific for immune effector precursors which are critical for an efficacious protection against viral reinfection. In fact, in our study, we have observed a higher amount of circulating anti-RSV antibodies with lower protecting activity in elderly persons. Early studies on influenza virus suggested that hypothetical suppressogenic epitopes can be responsible for induction of antigen-specific T "suppressor " cells . Why these regulatory cells exert their action on specific cells is still unclear and needs further study. In light of these results, manipulation of Tregs may be critical to enhance immune responses in the aged and new strategies aimed at limiting Tregs would be useful for prevention of severe RSV infection in frail, susceptible people.