In this manuscript, we provide evidence that zinc functions as an ionic signaling molecule after T cell activation. Cytoplasmic zinc concentrations increased within 1 min after TCR triggering as the result of an influx via the zinc transporter Zip6. The increase was most pronounced in the immediate subsynaptic area and enhanced TCR signaling, at least in part as a result of inhibition of SHP-1 recruitment. Consequently, TCR activation thresholds were lowered and T cell responses were induced under suboptimal conditions.
Zinc’s function as a novel second messenger/signaling ion has been predicted to be of similar importance to the well established role of calcium ions (Frederickson et al., 2005
; Rink and Haase, 2007
). Two different categories of intracellular zinc signaling have been identified. The first one depends on transcriptional changes in zinc transporter expression. This mechanism has been described in DC after LPS stimulation, which induces the expression of zinc exporters, lowers cytoplasmic zinc, and facilitates DC maturation (Kitamura et al., 2006
). A similar mechanism is also functional in T cells where increased expression of zinc importers 48–72 h after TCR stimulation increases cytoplasmic zinc concentrations. Zinc influx mediated by increased transcription of Zip6 induces the expression of MTs, which supports T cell proliferation and is of particular importance for T cell survival and expansion in the elderly (Lee et al., 2008
). Increased expression of Zip8 after T cell stimulation induces increased release of lysosomal zinc blocking calcineurin activity and increasing IFN-γ transcription (Aydemir et al., 2009
). In contrast, a direct effect of cell activation on zinc homeostasis has been shown for mast cells, where cross-linking of the high-affinity IgE receptor induces release of free zinc from the perinuclear area (Yamasaki et al., 2007
). This increase in cytoplasmic zinc was coming from intracellular sources and depended on a calcium signal as well as activation of the Ras–MAPK pathway. Also, Kaltenberg et al. (2010)
showed that activation of the ERK pathway after IL-2 receptor stimulation was zinc dependent, presumably as a result of inhibition of phosphatase activity. In our study, a change in intracellular zinc concentration was observed within 1 min after T cell activation, documenting a direct effect on transporter activity. In contrast to the study by Yamasaki et al. (2007)
, the increase in cytoplasmic zinc was dependent on extracellular sources and correlated to the concentration of zinc in the medium. The increase in cytoplasmic zinc was largely prevented by depleting zinc from the medium or by silencing the Zip6 transporter that is expressed in the cytoplasmic membrane.
Surprisingly, the increased zinc concentrations were mostly confined to the subsynaptic area of the T cell–DC interaction platform ( and ). This pattern is in stark contrast to the activation-induced influx of calcium ions, where waves of increased calcium concentrations readily spread throughout the cell. One possible explanation for this pattern is that TCR activation induces clustering and activation of Zip6 transporters in the synaptic region consistent with the dependency of subsynaptic zinc accumulation on extracellular zinc concentrations. However, a contribution from intracellular sources cannot be excluded. For calcium, release from intracellular stores is required for propagating and sustaining the signal. In activated T cells, zinc is released from lysosomes via a Zip8-dependent mechanism. Zip8 is expressed in resting T cells, although it is largely up-regulated after activation, a pattern which it shares with Zip6. Staining with Lysotracker showed accumulation of lysosomes in the subsynaptic region colocalizing with increased FluoZin-3 fluorescence (unpublished data). In this model, Zip6-dependent zinc influx from extracellular sources after T cell activation cooperates with redistribution of lysosomes and zinc release to build a steep gradient of zinc concentration in the subsynaptic region that influences proximal TCR signaling.
Early events in TCR signaling are tyrosine phosphorylations of several signaling molecules. The src protein kinase Lck is primarily responsible for the early phosphorylation events of tyrosines within the ITAM motifs of CD3ζ and ZAP70 (Palacios and Weiss, 2004
). Data in show that extracellular zinc influences ZAP70 phosphorylation. Lck is bound to CD4 or CD8. In fact, the sequestration of Lck by CD4 or CD8 has been identified as the major mechanism imposing a MHC class I or II restriction on T cell responses (Van Laethem et al., 2007
). This binding of Lck to CD4 or CD8 is dependent on zinc, suggesting that zinc functions as a structural element in synapse formation. However, our studies did not confirm the hypothesis that zinc influx is important in CD4-Lck binding. Immune precipitation studies of the TCR activation complex did not demonstrate an influence of extracellular zinc concentrations on Lck recruitment to the TCR synapses ().
Our studies show that inhibition of negative regulatory feedback loops account at least in part for the increased ZAP70 phosphorylation. A prime candidate is SHP-1, which has been shown to be recruited to Lck and then to dephosphorylate tyrosines within ITAM motifs of ZAP70 and other signaling molecules (Altan-Bonnet and Germain, 2005
). In our immune precipitation studies, recruitment of SHP-1 to the TCR synapse was reduced by increasing zinc influx. It is possible that zinc induces steric conformation changes in SHP-1 that prevent binding to Lck. Alternatively, increased ERK activity as a result of phosphatase inhibition may cause serine phosphorylation of Lck that prevents SHP-1 binding. We and others have shown that the dual-specific phosphatase DUSP6 is involved in TCR threshold calibration by increasing ERK activity (Li et al., 2007
), and DUSP activity has been shown to be sensitive to zinc concentrations (Junttila et al., 2008
It cannot be excluded that in addition to reduced SHP-1 recruitment, direct modulation of phosphatase or kinase enzyme activities by zinc also contributes to the increased signaling. Supplementation of cell lysates with 5–20 µM zinc yielded free zinc concentrations in the physiological nanomolar range and dose-dependently suppressed total phosphatase activity (Kaltenberg et al., 2010
), suggesting that zinc fluctuations in space and time regulate phosphorylation signals. In vitro, SHP-1 has an IC50 of 93 nM (Haase and Maret, 2003
), which may be achieved by the local gradient in the subsynaptic region. In addition, zinc may influence the regulation of Lck activity by phosphorylation of inhibitory tyrosines in the C terminus at position Y505 (Mustelin and Burn, 1993
). The balance between active and inactive Lck is maintained by the PTP CD45 and the kinase Csk. Whereas CD45 dephosphorylates Lck Y505, Csk rephosphorylates Lck at this position (Mustelin et al., 2005
). Activity of Csk is regulated by two mechanisms. The compartmentalization of Csk is determined by its binding protein that recruits Csk to the cell membrane (Rahmouni et al., 2005
). Upon T cell activation, Csk binds to the phosphoprotein G3bp, which is situated in an intracellular location adjacent to the immune synapse and, therefore, is presumed to exclude Csk from the initial signaling complex. Lck phosphorylates the cell membrane protein Cbp/PAG, which then recruits Csk to the synapse leading to the inactivation of Lck. Csk kinase activity has been shown to be magnesium dependent and inhibited by zinc (Sun and Budde, 1999
). This inhibition occurs in nanomolar range and is reversible. In addition to its phosphorylation of the C terminus–negative regulatory tyrosine, Csk also affects Lck activity by recruiting PTPN22, which dephosphorylates the positive regulatory tyrosines on Lck. PTPs have been shown to be particularly sensitive to the inhibitory action of zinc ion (Haase and Maret, 2005
). The classical example is PTP1B, which is inhibited by zinc in vitro at an IC50 of 17 nM, a concentration which is likely to be achieved in vivo. Data on the zinc sensitivity of PTPN22 are not available, but inhibition of both Csk and PTPN22 could contribute to the sustained Lck activation that we have observed with increased zinc influx.
Zinc influx was seen in in vitro systems that closely mimicked physiological activation. We used a DC–superantigen system to stimulate peripheral blood CD4 T cell, which allowed us to study the influx of zinc and signaling events in the context of TCR synapse formation. If T cells were activated by pharmacological stimulation such as CD3/CD28 cross-linking, zinc influx and associated signaling modifications were less evident, emphasizing the importance of physiological synapse formation (unpublished data). We used a large range of zinc concentrations from 3 µM to supraphysiological concentrations of 45 and 75 µM in RPMI supplemented with 10% serum. Bioavailable free zinc linearly correlated with total zinc in our culture system over the entire dose range (Fig. S1
). Physiological zinc serum concentrations are ~15 µM, and zinc deficiency is defined as a serum level of <10.7 µM (Lichten and Cousins, 2009
). The relevant zinc concentrations in biological compartments are unknown, and serum zinc concentrations are poor biomarkers of the zinc status (Lichten and Cousins, 2009
). In all of our assay systems, results directly correlated with zinc concentrations in the medium, and effects were consistently seen at <30 µM, suggesting that activation-induced ionic zinc signaling is physiologically relevant. In most experiments, effects leveled off at concentrations of >30–45 µM zinc.
In the functional studies, increased zinc influx lowered the threshold to TCR stimulation, which is consistent with the concept that the local cytoplasmic accumulation of zinc inhibits a negative-feedback mechanism in TCR signaling. Proliferative T cell responses under suboptimal antigen conditions were improved by higher media concentration of zinc. We used several systems to examine this hypothesis. Results in all systems were consistent. Higher extracellular zinc concentrations increased the proliferative responses to suboptimal anti-CD3 stimulation. In the more physiological system, we used naive T cells, mDCs, and the superantigen TSST. Under normal conditions, TSST only activates Vβ2+
cells. However, under optimized conditions, small numbers of Vβ2−
T cells, which presumably have a low affinity to TSST, are also activated by the superantigen. Increased medium concentration of zinc allowed for the stimulation of Vβ2−
T cells, suggesting that zinc facilitates a low avidity T cell response. Finally, increased concentrations could compensate for lower antigen peptide concentrations in an antigen-specific response using naive T cells from TCR transgenic mouse and the appropriate peptide antigen. This increased responsiveness is explained by a decreased recruitment of SHP-1 to the TCR activation complex. Interestingly, increased zinc concentrations could not restore the immune response to an antagonistic peptide (unpublished data), although the negative regulatory feedback mechanism by SHP-1 has been implicated in the tolerance induction by antagonistic peptides (Altan-Bonnet and Germain, 2005
). Apparently, zinc influx is sufficient to improve TCR signaling but not to a degree that interferes with tolerance mechanisms.
We propose a model where influx of zinc after TCR stimulation leads to a local increase in cytoplasmic zinc, modifies early TCR signaling events, and selectively lowers TCR activation thresholds. The local confinement of increased zinc concentrations targets the effect to amplifying proximal TCR signals without globally modifying the many different cellular processes regulated by zinc-binding proteins. Because the zinc influx originates from extracellular sources, local manipulation of zinc availability may be a means to enhance T cell responses to antigens or vaccines. A target of particular interest is the zinc importer Zip6, which accounts for the zinc influx during T cell activation.