Po et al. demonstrate that the specific CD8 T cell response to primary influenza infection is altered in aged mice, as shown by decreased and delayed CD8 T cell expansion as well as specific CD8 T cell function as assessed by IFN-γ production (Po, Gardner et al. 2002
). While many studies link the age-altered changes in T cells to intrinsic defects, we hypothesized that the extrinsic environment of aged mice may also contribute to the decrease. Specifically we hypothesized that this decreased CD8 T cell expansion and function may be linked to the increase in Treg cells seen in aged mice.
Since Treg cell percentage is increased in aged mice (Nishioka, Nishida et al. 2008
), we wanted to study the effects of influenza infection on this population of CD4 T cells. As early as 5 days after infection we see a significant increase in the percentage of Treg cells in aged mice while young mice maintain a similar percentage of Treg cells throughout the course of infection (). The expansion of Treg cells in aged mice could provide a potential hurdle for the immune response to overcome, as other studies have demonstrated Treg cells can interfere with the in vivo
immune response (Zelinskyy, Dietze et al. 2009
). It is not until day 10 when the percentage of Treg cells in aged mice returns to basal level that the CD8 T cells begin to expand and respond to influenza infection by producing IFN-γ. Interestingly after influenza infection, Treg cells have an increase state of activation as demonstrated by increasing percentages and increased expression of CD69 both before and during infection. We hypothesize that this early and continued expansion of Treg cells in aged mice may interfere with expansion and function of specific CD8 T cell.
Several studies have compared the percentages of Treg cells. As previously discussed Treg cells have been defined differently based on their surface phenotype as being: CD4+
. In this report we are able to conclude that Treg cells are significantly increased in aged (>18 months) compared to young (4–6 months) mice in all three of the previously defined populations (). Han et al. proposed that a new subset of Treg cells, CD4+
, were also able to suppress effector T cell function (Han, Guo et al. 2009
). We, therefore, examined this subpopulation of CD4 T cells. We found that total CD4+
T cells as well as CD4+
Treg cells are also increased in aged mice compared to young (young vs aged, 8.9 ± 1.6% vs 19.4 ± 2.3%). These results provide an interesting set of data which shows that the regulatory component of the immune response is significantly changed in aging.
While there is clearly an increase in Treg cells with age, the question is whether their increased percentage alone or in conjunction with enhanced suppressive function contribute to the age-associated decline in response. Our results from in vitro
suppression assays agree with those of Sharma et al. and Nishioka et al. which suggest that there is no difference in per cell function of Treg cells of aged compared to young mice (Nishioka, Shimizu et al. 2006
; Sharma, Dominguez et al. 2006
). In addition, we show that Treg cells suppress CD8 T cell activation in a dose dependent manner (Figure 8). The dose dependent suppression of effector function, demonstrated by the significant R2
values seen in correlation studies, is critical when there is increasing percentages found in aged mice both prior to and during and immune response to influenza infection. This increased percentage of Treg cells may raise the threshold of activation required to produce a strong immune response to foreign antigen.
Although we report no significant difference in the suppressive function of Treg cells in a suppression assay, our results from MFI shows that aged mice have an increased expression of GITR, whose ligand is found on APCs. The GITR ligand usually binds to T cells during an immune response and this binding enhances antigen presentation as well as activation of APCs and T cells. The constitutive expression of GITR on Treg cells allows them to bind to APCs as well and to interfere with antigen presentation and T cell activation. It has been reported that G3c, a mAb which recognizes GITR, breaks the hypoproliferative state of Treg cells and cause them to expand both in vivo
and in vitro
(Nishioka, Nishida et al. 2008
). Therefore we postulate that binding of Treg cells to APCs via GITR can both down-regulate an immune response by interfering with antigen presentation and can also lead to the expansion of Treg cells. GITR should be further analyzed as a mechanism, which leads to the expansion of Treg cells following influenza infection in aged mice.
The results of this study strongly suggest that the presence of increasing percentages of activated Treg cells in aged mice is a contributing factor in the decreased and delayed CD8 T cell during primary influenza infection.
- Upon influenza infection, aged, but not young, mice have a significant expansion of Treg cells.
- Treg cells of aged mice demonstrate both a higher percentage and higher expression per cell of CD69 both at baseline and during infection compared to young mice.
- Treg cells from young and aged mice comparably suppress CD8 T cells and suppression is dose dependent.
- These results suggest that the increase in the percentage of Treg cells in aged mice may contribute to the diminished CD8 T cell response to primary influenza infection.