While transcriptionally profiling naïve CD4 T cell responses in the adult, we identified a hereto undefined molecular pathway that is important to T cell activation and that is modified with age. Antigen-mediated stimulation of naïve CD4 T cells transiently increased cytoplasmic labile zinc, at least in part through the zinc importer Zip6. The increase in cytoplasmic labile zinc induces the transcription of MT in a negative feedback loop. MT influence the intracellular milieu through several mechanisms, including their ability to scavenge ROS and to control zinc delivery to metalloproteins. The activity of this pathway is more pronounced with age; naïve CD4 T cells from young adults have a less sustained cytoplasmic labile zinc elevation and consequently lower MT expression after stimulation than older individuals.
We focused on the age period between 60 and 75 years because the age of 75 years appears to be a turning point for naïve CD4 cells, after which the repertoire rapidly contracts and naïve cells are increasingly defective.28
The naïve CD4 T cell response was assessed in an in vitro
system where T cell priming was simulated by using a superantigen and mDC. Superantigen concentration and the number of mDC were chosen to be suboptimal to unmask putative age-dependent differences. Global T cell responsiveness was intact, as documented in the functional assays as well as in the transcriptome studies, suggesting that TCR-mediated signaling and costimulation were not affected in this age group. Surprisingly, the functional studies documented a higher proliferative rate in naïve CD4 T cells in the older age group. This finding was counterintuitive because human naïve CD4 T cells with age show evidence of an extensive replicative history with telomeric erosion.29
T cell stimulation was associated with an impressive up-regulation of the MT family members MT1E, F, G, H, M, X, and MT2A in young adults and more so in individuals aged 60 years and older. In kinetic studies, MT transcriptional activity peaked later than traditional T cell activation markers, raising the possibility of an intermediary mechanism controlling the T cell activation-induced transcription. MT synthesis is primarily controlled by zinc that enables MTF-1 to bind to the multiple metal response elements in the MT promoter.30,31
In addition to zinc, a number of inflammatory cytokines, including interferon-γ, TNF-α, and IL-6, induce MT transcription. Gene expression profiling did not yield any age-dependent fingerprint in these cytokine genes, and therefore we focused on the hypothesis that age influences cellular zinc homeostasis.
Bioavailable cytoplasmic zinc in stimulated CD4 T cells increased in the first 48–72 h and then slowly returned back to prestimulation levels. This decline was blunted in activated CD4 naïve T cells from 60- to 75-year-old individuals. Cellular components that regulate the cellular zinc homeostasis are MT and zinc transporters.30,32,33
MT bind up to seven zinc atoms via a total of 20 cysteines. This binding is redox sensitive; increased oxidative or nitrosative stress can reduce the binding capacity of MT and release zinc.34,35
Data shown in clearly demonstrate that MT is an important redox system in activated T cells. Therefore, increased production of ROS during T cell activation in the elderly could explain increased zinc bioavailability and MT induction. Alternatively, zinc transport mechanisms may be involved. Two zinc transporter systems can be distinguished.33,36,37
Members of the ZnT (SLC30A) family lower intracellular zinc by mediating zinc efflux into the extracellular fluid or influx into intracellular vesicles.37
To the contrary, Zip proteins of the SLC39A family mediate the influx of zinc from extracellular or intracellular sources into the cytoplasm.36
Effective negative feedback loops with induction of MT or ZnT molecules explain the normalization of cytoplasmic labile zinc in activated T cells, a feedback loop that was obviously even more induced in the elderly. The increased concentration of cytoplasmic labile zinc that we observed in activated T cells was dependent on Zip6 transporter expression. Silencing of Zip6 significantly reduced MT expression after T cell stimulation, confirming that the activation-induced upregulation of MT is due to zinc influx. The increased MT expression in stimulated naïve CD4 T cells of the older individuals could therefore be explained by increased Zip6 activity.
Fluctuations of intracellular zinc after stimulation have only recently received attention.38
Mast cell activation via the Fc
RI receptor induces a zinc wave within minutes, presumably through increased activity of intracellular zinc transporters.39
In contrast, DC respond to lipopolysaccharide stimulation with a change in the expression profile of zinc transporters and decrease in cytoplasmic labile zinc, which is necessary for DC maturation.40
In our studies, T cells respond to antigenic stimulation with the activation of the zinc importer Zip6 and the influx of zinc from extracellular sources. The increased intracellular level of labile zinc results in the transcriptional activation of a selected set of genes including MT and ZnT1, which lower the level of cytoplasmic labile zinc again.
Why T cells respond to antigen stimulation with zinc influx and a manifold MT induction and what the functional consequences are of the increased expression in individuals older than 60 years is of particular interest. Zinc is involved in many biological processes.41–43
The immune system is particularly dependent on zinc,44
and zinc deficiency has been implicated in the declining immune function with age,45,46
MT function in zinc homeostasis, protection against exogenous heavy metal toxicity, and intrinsic oxidative damage.47
The higher zinc influx and the associated induction of MT may represent a protective mechanism for the aging T cell to neutralize the potentially damaging effects of ROS produced during activation and to increase the proliferative potential, which is of utmost importance for the rapid clonal expansion upon antigen recognition that is characteristic of successful T cell immunity. Indeed, there was a strict correlation between activation-induced proliferation and MT expression, and silencing of MT expression significantly increased apoptosis and reduced cell recovery.