Research ArticleImmunology

Human Regulatory T Cells Rapidly Suppress T Cell Receptor–Induced Ca2+, NF-κB, and NFAT Signaling in Conventional T Cells

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Science Signaling  20 Dec 2011:
Vol. 4, Issue 204, pp. ra90
DOI: 10.1126/scisignal.2002179
  • Fig. 1

    Suppression of Tcons persists after the removal of Tregs. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of the abundances of IL2 and IFNg mRNAs in HLA-A2 Tcons. (A) HLA-A2 Tcons were cocultured with pre-activated HLA-A2+ Tregs (supTcon) or with HLA-A2+ Tcons as a control (cTcon) at a 1:1 ratio and stimulated with cross-linked antibodies against CD3 and CD28 for 3 hours. HLA-A2 Tcons were then isolated and the abundances of IL2 and IFNg mRNAs were analyzed. Results are presented as the fold change in the abundance of the indicated mRNA in stimulated suppressed Tcons or stimulated control Tcons compared to that in unstimulated Tcons, which was set to 1. Data are means ± SD of duplicate samples of a single donor and are representative of >20 donors. The percentages of reduction in the abundances of IL2 and IFNg mRNAs in the suppressed Tcons compared to those in control Tcons are indicated in gray (% Suppression). (B) Cells were cocultured as described for (A) (coculture stimulation, left bars) or were cocultured for 10 to 60 min before undergoing stimulation of isolated responder Tcons (“pre-cocultured”). For the pre-cocultures, HLA-A2 Tcons were left unstimulated in the presence of pre-activated Tregs or Tcons, as a control, at a 1:1 ratio. HLA-A2 Tcons were then isolated and stimulated alone as described in (A), and the abundances of mRNAs for the indicated cytokines were measured. Shown are the percentages of suppression as described for (A). n.s., not significant. (C) Cocultures were established as described in (B), with the exception that after a pre-coculture period of 60 min, HLA-A2 Tcons were isolated and rested for 24 hours before they were stimulated. Data are the means ± SEM from four (B) or three (C) donors. Statistical significance of suppression was calculated with the Student’s one-sample t test.

  • Fig. 2

    NF-κB signaling is inhibited in suppressed Tcons. (A to C) Western blotting analysis of the phosphorylation of (A) PKCθ (pT538), (B) IKKα (pS180) and IKKβ (pS181), and (C) IκBα (pS32) in HLA-A2 Tcons. Cocultures were established as described for Fig. 1A, and cells were stimulated for the indicated times. Suppressed Tcons or control Tcons were analyzed with phosphospecific antibodies (upper images in each panel). Blots were then incubated with antibodies against the indicated total proteins and with antibody against tubulin, which served as a loading control. Blots are from one experiment representative of 4 (A), 3 (B), or >12 (C) donors. The right panel in (C) shows the mean percentage suppression of IκBα phosphorylation ± SEM from 19 (5 min) or 12 (25 min) donors. Statistical significance of suppression was calculated with the Student’s one-sample t test. (D) Western blotting analysis of the amount of phosphorylated p65 (p-p65). Suppressed Tcons (red) or control Tcons (black) were analyzed with fluorescently labeled antibodies against p-p65 (pS536) and β-actin. One representative experiment from five different donors is shown. (E) NF-κB target genes obtained from gene array data. The gene expression patterns of control and suppressed Tcons were compared after 3 hours of TCR stimulation. D signifies genes whose expression was significantly decreased in suppressed Tcons compared to that in control Tcons. The array was performed with two different donors, and one donor is shown. T, threonine; S, serine.

  • Fig. 3

    NFAT1 dephosphorylation is inhibited in suppressed Tcons. (A) Western blotting analysis of NFAT1 dephosphorylation in HLA-A2 Tcons. Cocultures were established as described for Fig. 1A and cells were stimulated for the indicated times. Suppressed Tcons and control Tcons were then analyzed for the presence of phosphorylated and total NFAT1 proteins. The upper bands represent phosphorylated NFAT1 (p-NFAT1), whereas the lower bands represent dephosphorylated NFAT1. Blots were then incubated with antibody against tubulin as a loading control. Data are from one donor representative of 11 (5 min) or 10 (25 min) donors. Lower graph shows the percentage suppression of NFAT1 dephosphorylation as determined by densitometric analysis of Western blots as shown in the upper panels. Data are the means ± SEM of 11 (5 min) or 10 (25 min) donors. Statistical significance of suppression was determined by Student’s one-sample t test. (B) NFAT target genes obtained from gene array analysis as described for Fig. 2E. D, genes whose expression in suppressed Tcons was significantly decreased compared to that in control Tcons; MD, marginal decrease.

  • Fig. 4

    AP-1 signaling is not involved in Treg-mediated suppression of Tcons. (A) Luminex analysis of the phosphorylation of ERK1 and ERK2 (ERK1/2) in HLA-A2 Tcons after TCR activation. Cocultures were established as described for Fig. 1A, and cells were stimulated for the indicated times. Suppressed Tcons and control Tcons were analyzed for the extent of phosphorylation of ERK1/2 (pT185/pY187). Values were normalized to account for the amounts of total protein. Data are expressed as the fold change in the abundances of the indicated phosphorylated protein compared to those in unstimulated control Tcons, which were set at 1. Analyses were performed in duplicate, and mean fold change ± SD is shown. Data are from one donor representative of three different donors. (B) Western blotting analysis of the presence of p-p38 (pT180/pY182) in suppressed Tcons and control Tcons. Blots were analyzed with antibody against p-p38 and then incubated with antibodies against total p38 and tubulin. Data are from one experiment that is representative of five experiments. (C) Analysis of the phosphorylation of c-Jun (pS63) as described for (A). Data are from one donor and are representative of five donors. (D) Analysis of the expression of AP-1 target genes obtained from gene array assays as described for Fig. 2E. NC, genes whose expression in suppressed Tcons was not significantly different from that in control Tcons; Y, tyrosine.

  • Fig. 5

    Ca2+ signaling is abrogated in suppressed Tcons independently of IP3. (A) Tcons were labeled with Indo-1 AM and PKH67 and cocultured with PKH26-labeled, pre-activated allogeneic Tregs (supTcon) or with allogeneic Tcons (cTcon) for at least 30 min. Cells were analyzed by flow cytometry and gated on Indo-loaded responder Tcons, as shown in the dot plots, and cells were then monitored for Ca2+ signaling as shown in the bottom panels depicting Ca2+ traces. After 1 min to establish a baseline, cross-linked antibodies against CD3 and CD28 were added to the cells (arrow) to stimulate Ca2+ signaling. The lower left panel shows Ca2+ influx in unconjugated Tcons (stained only with PKH67, from the Q4 gate in the upper dot plot), whereas the lower right panel shows Ca2+ influx in conjugated Tcons (PKH67- and PKH26-positive cells, from the Q2 gate in the upper dot plot). Data are from one donor and are representative of 20 donors. (B) Cells were labeled and cocultured as described for (A). Cocultured cells were then washed, resuspended in Ca2+-free PBS, and analyzed for Ca2+ signaling. After 1 min, cross-linked antibodies against CD3 and CD28 were added to the cells (arrow), and then medium was added to provide a final extracellular concentration of Ca2+ of 0.75 mM (arrowhead). Ca2+ influx in unconjugated Tcons (from the Q4 gate) is shown. Data are from one donor representative of seven independent donors. (C) Responder Tcons were labeled with [3H]inositol, cocultured with either unlabeled Tcons (cTcon) or unlabeled Tregs (supTcon), and then were stimulated for 5 min or were left unstimulated. Inositol phosphates were extracted from the cells and fractionated by HPLC, and the counts per minute (cpm) values were measured. Data are from one donor and are representative of five donors. OD, optical density.

  • Fig. 6

    Forced depletion of intracellular Ca2+ stores abrogates Treg-mediated inhibition of Ca2+, NFAT, and NF-κB signals in Tcons. (A to C) Cells were labeled and cocultured as described for Fig. 5. (A) Analysis in Ca2+-free PBS. The arrow indicates the addition of cross-linked antibodies against CD3 and CD28 in the absence or presence of thapsigargin (TG, 1 μM). The arrowheads at the top of the graph indicate the addition of medium to obtain an extracellular Ca2+ concentration of 0.5 mM. (B and C) The arrow indicates the addition of cross-linked antibodies against CD3 and CD28. Dashed arrow indicates the addition of (B) 100 nM or (C) 1 μM thapsigargin. Ca2+ influx in conjugated Tcons is shown. Data are from a single donor representative of four (A) or three (B and C) donors. (D) HLA-A2 Tcons were cocultured with HLA-A2+ Tcons (cTcon) or pre-activated HLA-A2+ Tregs (supTcon) for 60 min, and then cell populations within the cocultures were separated on the basis of HLA-A2 expression. Middle panel shows Western blotting analysis of HLA-A2 Tcons stimulated with cross-linked antibodies against CD3 and CD28 in the absence or presence of ionomycin (500 nM) or thapsigargin (50 nM) for 5 min. Lower panel shows analysis of the abundance of IL2 mRNA in HLA-A2 Tcons after 2 hours of stimulation with cross-linked antibodies against CD3 and CD28 in the absence or presence of ionomycin (100 and 500 nM). Relative mRNA amounts were normalized to those of GAPDH mRNA. Results are presented as the fold change in the abundance of IL2 mRNA compared to that in unstimulated Tcons, which was set to 1. Data are the means ± SD of the analysis of duplicate samples from one donor by qRT-PCR and are representative of three donors.

Additional Files

  • Supplementary Materials for:

    Human Regulatory T Cells Rapidly Suppress T Cell Receptor–Induced Ca2+, NF-κB, and NFAT Signaling in Conventional T Cells

    Angelika Schmidt, Nina Oberle, Eva-Maria Weiß, Diana Vobis, Stefan Frischbutter, Ria Baumgrass, Christine S. Falk, Mathias Haag, Britta Brügger, Hongying Lin, Georg W. Mayr, Peter Reichardt, Matthias Gunzer, Elisabeth Suri-Payer, Peter H. Krammer*

    *To whom correspondence should be addressed. E-mail: P.Krammer{at}dkfz.de

    This PDF file includes:

    • Methods
    • Fig. S1. Suppression of cytokine secretion is retained after removal of Tregs.
    • Fig. S2. Treg-mediated rapid suppression of IL2 expression in Tcons also occurs in the presence of APCs.
    • Fig. S3. CTLA-4 is not involved in the suppression of cytokine gene expression.
    • Fig. S4. Proximal TCR signaling is not altered in suppressed Tcons.
    • Fig. S5. Suppression of NFAT and NF-κB activation is independent of the antibody used to activate Tregs.
    • Fig. S6. Phosphorylation of ERK and p38 is not affected in suppressed Tcons.
    • Fig. S7. The block in Ca2+ influx in Tcons is not caused by allogeneic responses and requires 30 min of previous coculture with Tregs.
    • Fig. S8. Suppression of Ca2+ signaling is independent of cAMP.
    • Fig. S9. Tregs, but not Tcons, pre-activated with covalently plate-bound antibody against CD3 suppress Ca2+ signaling in responder Tcons.
    • Fig. S10. NFAT and NF-κB activation in Tcons is Ca2+-dependent.
    • References

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    Technical Details

    Format: Adobe Acrobat PDF

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    Citation: A. Schmidt, N. Oberle, E.-M. Weiß, D. Vobis, S. Frischbutter, R. Baumgrass, C. S. Falk, M. Haag, B. Brügger, H. Lin, G. W. Mayr, P. Reichardt, M. Gunzer, E. Suri-Payer, P.H. Krammer, Human Regulatory T Cells Rapidly Suppress T Cell Receptor–Induced Ca2+, NF-κB, and NFAT Signaling in Conventional T Cells. Sci. Signal. 4, ra90 (2011).

    © 2011 American Association for the Advancement of Science

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