Zap70 Regulates TCR-Mediated Zip6 Activation at the Immunological Synapse

Abstract

The essential microelement zinc plays immunoregulatory roles via its ability to influence signaling pathways. Zinc deficiency impairs overall immune function and resultantly increases susceptibility to infection. Thus, zinc is considered as an immune-boosting supplement for populations with hypozincemia at high-risk for infection. Besides its role as a structural cofactor of many proteins, zinc also acts as an intracellular messenger in immune cell signaling. T-cell activation instructs zinc influx from extracellular and subcellular sources through the Zip6 and Zip8 zinc transporters, respectively. Increased cytoplasmic zinc participates in the regulation of T-cell responses by modifying activation signaling. However, the mechanism underlying the activation-dependent movement of zinc ions by Zip transporters in T cells remains elusive. Here, we demonstrate that Zip6, one of the most abundantly expressed Zip transporters in T cells, is mainly localized to lipid rafts in human T cells and is recruited into the immunological synapse in response to TCR stimulation. This was demonstrated through confocal imaging of the interaction between CD4⁺ T cells and antigen-presenting cells. Further, immunoprecipitation assays show that TCR triggering induces tyrosine phosphorylation of Zip6, which has at least three putative tyrosine motifs in its long cytoplasmic region, and this phosphorylation is coupled with its physical interaction with Zap70. Silencing Zip6 reduces zinc influx from extracellular sources and suppresses T-cell responses, suggesting an interaction between Zip6-mediated zinc influx and TCR activation. These results provide new insights into the mechanism through which Zip6-mediated zinc influx occurs in a TCR activation-dependent manner in human CD4⁺ T cells.

Keywords: T cell receptor (TCR); T lymphocytes; Zip6; immunological synapse; lipid rafts; zinc.

Figure 1

Zip6 is predominantly localized in lipid rafts in human CD4⁺ T cells. Jurkat cells (A) and human primary CD4⁺ T cells (B) were unstimulated (Left) or stimulated with anti-CD3/CD28 monoclonal antibodies (mAbs) for 15 min at 37°C (Right). Cells were lysed in 1% Triton X-100 buffer and subjected to sucrose density gradients for isolation of the lipid raft fractions. (A) Twelve aliquots of fractions collected from the top of the density gradients of unstimulated (Left) or stimulated (Right) Jurkat cell lysates were analyzed by dot blot analysis for GM1, a marker of lipid rafts, and CD71, a marker of non-lipid raft fractions, to identify detergent-resistant membrane fractions containing lipid rafts (Fractions 3-6). Twelve aliquots were further separated by SDS-PAGE and immunoblotted for Lck and Flot-1, additional markers of lipid rafts, and Zip6. (B) Twelve aliquots of fractions collected from unstimulated (Left) or stimulated (Right) human primary CD4⁺ T cell lysates were examined by immunoblotting forZip6, Flot-1, and Lck. Data is a representative of three independent experiments with cell lines at different passages or three different healthy donors.

Figure 2

Zip6 is recruited into the immunological synapse of stimulated T cells. (A) Monocyte-derived DCs were loaded with or without SEB and TSST-1 (500 ng/ml) for 30 min and pelleted with primary CD4⁺ T cells by centrifugation followed by incubation for 30 min at 37°C. Cells were allowed to adhere to poly-lysine coated glass slides, and fixed, permeabilized and stained for CD3 (red) and Zip6 (green), and analyzed by confocal microscopy. Arrow indicates the accumulation of Zip6 and CD3 at the established immunological synaptic area. (B) Human CD4⁺ T cells were pelleted with or without anti-CD3/28 antibody-coated microbeads by centrifugation, followed by incubation for 30 min at 37°C. Cells were fixed, permeabilized, and stained with anti-Zip6 (green) and CD3 (red) antibodies, and analyzed by confocal microscopy. Accumulation of Zip6 (green) and CD3 (red) was quantified by fluorescence along a cross section perpendicular to the synapse (white dotted line arrow in left panel) as line-intensity histograms (middle panel). Fluorescence intensity of CD3 and Zip6 at the site of T cell-microbeads interaction was dramatically increased in human CD4⁺ T cells (right panel). Results are from three independent experiments. Scale bars represent 5 μm. Bar graphs show the mean ± S.E.M. **** = p < 0.0001 by two-tailed paired t-test.

Figure 3

Zip6 is phosphorylated by Zap70 in response to TCR stimulation. Jurkat cells were coated with anti-CD3 and anti-CD28 Abs, followed by cross-linking with goat anti-mouse IgG for the indicated times and under the indicated conditions at 37°C. The lysates were prepared and immunoprecipitated with our in-house anti-human Zip6 Ab (hZip6) or control IgG. (A) Immunoprecipitates (IP) were analyzed by immunoblot analysis to detect tyrosine phosphorylation of Zip6. Data are representative of experiments replicated at least three times. Red arrows indicate target bands (B) Jurkat cells were stimulated, lysed, and immunoprecipitated with hZip6 Ab or control IgG as described in (A). Immunoprecipitates were analyzed by immunoblot analysis to detect Lck and Zap70. Data are representative of three experiments. (C) Jurkat cells were stimulated by CD3/CD28 cross-linking for 30 min in the presence of Lck inhibitor (RK-24466; 100 nM) to block phosphorylation of Zap70, lysed and immunoprecipitated with hZip6 Ab or control IgG as described in (A). Immunoprecipitates were analyzed by immunoblot analysis to detect Zap70. Data are representative of three experiments. (D) Jurkat cells were pre-treated for 30 min with the indicated concentration of Zap70 inhibitor (Zap180013), followed by stimulation with CD3/CD28 cross-linking for 5 min. Cell lysate were prepared for immunoblotting. (E) Jurkat cells pretreated with or without 4 μM of Zap180013 were stimulated with CD3/CD28 cross-linking for 15 min, lysed, and immunoprecipitated with hZip6 Ab or control IgG. Immunoprecipitates were analyzed by immunoblot analysis to detect phospho-tyrosine.

Figure 4

Zip6-mediated zinc influx is induced by TCR activation in CD4⁺ T cells. Freshly purified human CD4⁺ T cells were coated with anti-CD3 and anti-CD28 Abs, followed by cross-linking with goat anti-mouse IgG for the indicated times and under the indicated conditions at 37°C (A) Metallothionein 2A (MT2A) mRNA was quantified by real-time RT-PCR at the indicated time points (n = 4). (B) Human CD4⁺ T cells were stimulated by crosslinking for 3 h in the presence of 1.5 μM TPEN (TP) or different concentrations of ZnCl₂ (0, 3, and 15 μM). MT2A mRNA level was quantified by real-time RT-PCR (n = 7). (C) Jurkat cells were pretreated with RK-24466, a Lck inhibitor for 30 min at 37°C, followed by stimulation by CD3/CD28 cross-linking for 30 min at 37°C. Cells were stained with FluoZin-3, a zinc-specific fluorescent probe and its fluorescent intensity was analyzed by flow cytometry. MFI is a value between stained cells versus non-stained cells. (D) Jurkat cells (left) and human CD4⁺ T cells (right) were transfected with scrambled control siRNA (siCon; black line bar) or Zip6 siRNA (siZip6; red line bar) for 48 h. Cells were pre-loaded with FluoZin-3, a zinc-specific fluorescent probe, and coated for 30 min with anti-CD3 and anti-CD28 Abs, followed by cross-linking with anti-mouse IgG for 30 min at 37°C (n = 6). Fluorescent intensity of FluoZin-3 was analyzed by flow cytometry. (E–G) Jurkat cells and CD4⁺ T cells transfected with control (siCon; black line bar) or Zip6 siRNA (siZip6; red line bar) were stimulated with anti-CD3/CD28 for 24 h (E) or 3h (F, G), followed by analysis of CD69 expression using flow cytometry or analysis of CD69, IL-2 and IFN-γ mRNA expression by real-time RT-PCR, respectively. Expression was normalized to β-actin, and the comparative Ct method was used for quantification of gene expression (A, B, F, G). Bar graphs show the mean ± S.E.M. *p < 0.05, **p < 0.01, and ****p < 0.0001 by two-tailed paired t-test.