To determine the effect of the aged environment on the Ag-specific CD8 T cell response to virus infection, the following questions were addressed in this study using two different genetic strains of mice (B6 and BALB/c mice) and two different viruses (influenza virus PR8 and LCMV Arm): 1) What is the difference in Ag-specific CD8 T cell responses to virus infection between young and aged mice? 2) Does the aged environment limit the clonal expansion of specific CD8 T cells after infection with virus? 3) Does the number and Ag presenting function of DCs change with aging?
Similar to our previous study of primary influenza infection in B6 mice (Po et al., 2002
), a decreased Ag-specific CD8 T cell response occurred in both BALB/c and B6 aged mice after infection with influenza virus. While many studies suggest that this decreased response is due to intrinsic changes in T cells, the contribution of extrinsic factors has not been established. Plowden et al (Plowden et al., 2004
) found that the Ag presenting function of macrophages of aged mice was defective in inducing Ag-driven naïve CD8 T cell clonal expansion, while Mittler et al (Mittler et al., 2004a
) reported that the aged lymphoid microenvironment inhibits memory, but not primary, CD4 T cell response. More recently, however, Tesar et al (Tesar et al., 2006
) showed that adoptively transferred virus-specific young CD8 T cells responded equally regardless of the host’s age, suggesting no age-associated alteration of the environment.
Due to the questions remaining regarding the ability of the aged environment to support T cell proliferation and function, we decided to explore this subject further. We chose to use CD8 T cells from TCR Tg mice in an adoptive transfer approach to explore whether the aged environment has an effect on Ag-specific CD8 T cell response to virus infection. Since TCR-Tg CD8 T cells exhibits high affinity to engage the epitope loaded in the MHC I molecule (Boulter et al., 2007
; Pitcher et al., 2003
), the expansion of the Tg T cells should be quicker and stronger than the virus-specific response of CD8 T cell of wt mice. Our results clearly demonstrate that some component(s) of the aged environment limit Ag-specific T cell response early after virus infection ( to ). While our data show this negative impact of the aged environment in two virus models (influenza and LCMV), in two genetic backgrounds (BALB/c and B6) mice, and using two doses of adoptively transferred Tg T cells suggesting a consistent observation, our data with the P14 model was in direct conflict with the results of Tesar et al (Tesar et al., 2006
) who reported no effect of the environment with a similar adoptive transfer model. Comparison of the two experiments revealed a major difference: although we both adoptively transferred large numbers of Tg CD8 T cells, we assessed response on Day 3 and they evaluated response on Day 8 after infection. We could not directly assess Day 7 in our P14 model, because we used the commercially available P14, Thy1.2 mice. Since the recipient B6 mice are also Thy1.2, we could not differentiate the response of donor vs recipient CD8 T cells on Day 7. Tesar et al were able to perform this assessment since they bred the P14 Tg onto a Thy1.1 background. However, to address the hypothesis that the difference between the two studies was due to the kinetics of the response, we utilized the Clone-4 system in which the Thy1.1/Thy1.2 distinction between responding CD8 T cells could be made. When higher numbers of Clone-4 cells were transferred, we found minimal expansion of Tg CD8 T cells in either young or aged recipients on Day 2 after infection (< 0.1% of total CD8 T cells were IFN-γ+
). We consistently found a significantly decreased expansion of the transferred Tg CD8 T cells in aged mice on Day 3 (Young vs aged: 23% vs 11%; ). By Day 7, the percent of CD8 T cells in young mice that were HA+
decreased to 9.7%, while aged mice demonstrate a smaller decrease to 9.3% HA+
. These results reflect the kinetics we previously reported after infection of wt mice in which young mice demonstrate an earlier peak of response. When the response is evaluated at the peak of response of the aged, the level of response appears comparable between young and aged mice, since the response of young had already contracted ( Po et al., 2002
). Although not a direct assessment of the difference between the results of Tesar et al and ourselves, these data in the Clone-4 system suggest that the lack of an age-associated difference observed by Tesar et al (Tesar et al., 2006
) could be due to the fact that the peak response in young had already contracted by Day 8, while the response of the aged mice had just reached its maximum. Our data in the Clone-4 system, using lower numbers of transferred Tg T cells, further support the interpretation that the difference in the two studies is due to kinetics. On Day 4 the response in young is 19 × 104
and increases tenfold to 18 × 105
on Day 7. In aged mice the response also increases tenfold from 4.8 × 104
to 6.8 × 105
on Day 4 to Day 7. The expansion of the lower numbers of specific Tg CD8 T cells was significantly decreased in aged compared to young mice on both Day 4 and Day 7, suggesting that with the lower numbers of Tg cells adoptively transferred the peak response in aged mice had not been reached by Day 7.
An interesting observation of the adoptive transfer model is seen in . Without infection, young and aged recipients demonstrate a comparable percentage of CFSE-labeled P14 cells. Upon infection, there is a proliferation of these virus-specific TCR Tg CD8 T cells resulting in a decreased percent of CFSE labeled cells. In young hosts, this proliferation results in an increase in virus-specific (GP+), but CFSE negative, CD8 T cells. However, in aged recipients, the percent of CFSE labeled CD8 T cells decreased with only a limited increase in virus-specific CFSE negative CD8 T cells. This loss of CD8 T cells that are responding to the virus suggests apoptosis maybe occurring due to the aged environment. The mechanism of this age-associated loss of virus-specific CD8 T cells requires further examination.
DCs play a pivotal role in generating both innate and adaptive immunity to infection (Lanzavecchia et al., 2001
; Sallusto et al., 2002
; Iwasaki et al., 2007
). However, studies regarding DC functions and aging are limited, and the results are conflicting. No consensus has been reached concerning the alteration of the number nor the function of DCs in aged mice or individuals (Tesar et al., 2006
; Linton et al., 2001
; Shurin et al., 2007
). When we used equal number of splenocytes as APCs, the priming of specific CD8 T cells in two virus systems is decreased in the presence of splenocytes from aged mice. This limited expansion in the presence of splenocytes of aged mice may be due to 1) decreased number of APCs; 2) decreased function of APCs; or 3) both. When equal numbers of purified DCs from young or aged mice were cultured with virus-specific CD8 T cells of young mice, the clonal proliferation and expansion of CD8 T cells were similar, indicating that the priming function of DCs from aged mice remains intact in vitro regardless of BALB/c or B6 background. This is supported by a previous study that showed that the functions of DCs from aged mice remain intact even though there exists slight differences in the percentages of myeloid vs lymphoid DCs and surface expression of MHC molecules in DCs between young and aged mice (Norian et al., 2004
). This suggested that the decreased expansion with comparable number of splenocytes was due to the number of DCs of aged mice. However, phenotypic analysis indicated that the percentage of DCs as defined by MHC II+
or by CD11b+
(LyDCs), and B220+
(pDCs) is comparable if not slightly higher in aged mice. The limited expansion of Tg CD8 T cells in the presence of comparable splenocytes suggests the presence of another cellular factor in aged mice that inhibits the proliferative response, possibly the increased percentage of CD4 Treg cells (Sharma et al., 2006
In addition to DCs, B cells and macrophages also have the ability to present Ag to T cells. It was reported that macrophages of aged mice exhibit defects in APC function resulting in impaired Ag-induced CD8 T cell clonal expansion in vitro (Plowden et al., 2004
). Few studies have been reported on the alteration of Ag presenting function of B cells with aging during virus infection. In a preliminary study we performed, purified B cells of aged mice were equally capable as B cells of young mice of supporting specific CD8 T cell expansion in vitro (data not shown).
The decrease in immunological function with aging is an important process in which multiple extrinsic and intrinsic factors may be involved. Although age-related alterations of T cell function have been extensively studied, the studies on the potential changes of host environment with aging are very limited. Our findings suggest that factors of the aged-microenvironment play an important role in the alteration of Ag-specific CD8 T cell response to virus infection. Further examination of the aged environment is needed in order to better understand the mechanisms of immune senescence with aging, and to improve the efficacy of immunization against viral infections in aged individuals.