Fig 2: Dynamics of phosphorylation and selected functional modules induced by TCR stimulation Dynamics and cluster distribution of regulated phosphotyrosines, phospho–serines, and phosphothreonines.Illustration of TCR-regulated phosphosites within the polycomb-repressive complex 1 (PRC1) (Ub: ubiquitination; Ac: acetylation). The corresponding log2-transformed MS intensities measured upon TCR activation for each of the biological replicates are shown below. Box plot elements: Center line corresponds to median, box limits correspond to the first and third quartiles, and whiskers indicate variability from Q1–1.5. IQR to Q3 + 1.5 IQR.Short-time TCR stimulation induces FOXO3 phosphorylation and NFATC2 dephosphorylation and promotes nucleus exit or entry, respectively. CD4+ T cells left unstimulated (-) or stimulated for 2 and 5 min with anti-CD3 plus anti-CD4 antibodies were subjected to nuclear/cytoplasmic fractionation before immunoblot analysis with antibodies specific for NFATC2 and FOXO3. Arrows indicate phosphorylation (P) and dephosphorylation (deP) forms of the transcription factors. Lamin-B2 and GAPDH are used to control purity of nuclear and cytoplasmic extracts.t-SNE plot highlighting phosphosites associated with the UniProt keywords “Protein biosynthesis” and “Translation regulation”. Dot transparency is scaled according to the P-value corresponding to the local enrichment of the annotation term (hypergeometric test, see method for more detailed information).Left: Dynamics of TCR-regulated phosphosites associated with the UniProt keywords “Protein biosynthesis” and “Translation regulation” (dots color-coded by cluster). Right: Schematic representation of the translational initiation and elongation complexes. TCR-regulated phosphosites are represented as small dots color-coded by cluster.

Fig 3: Tregs suppress CTL-mediated cytotoxicity by downregulating intracellular calcium signals.(a) B16F10 melanoma cells were intradermally injected into the ear skin. Tumour growth (bar) and Treg frequency (dot) were monitored. n>5 mice per group. (b) Phenotypic characteristics of tumour infiltrating CD4+Foxp3+ Treg cells. (c) CTL-mediated tumour killing assay. OT-I CTLs were incubated for 5 h with OVA peptide-loaded EL-4 cells at a 1:1 ratio in the presence of rTregs or aTregs. Apoptotic cells were stained for Annexin V (n=3). (d,e) Expression of perforin, granzyme B and CD107a on OT-I CTLs incubated with OVA-pulsed EL-4 at a 1:1 ratio in the absence or presence of aTregs was measure by flow cytometry (n=3). (f) Schematic for intracellular Ca2+ signals in CTL. Stimulation of TCR triggers Inositol 1,4,5 trisphosphate (IP3) production via Zap70 and PLC? activation. IP3 binds to the IP3 receptor (IP3 Rc) on the ER membrane to empty the ER Ca2+ store. ER Ca2+ depletion induces STIM1 oligomerization leading to CRAC channel (Orai1) opening and (Ca2+)i increase. (g) Western blot analysis of TCR signaling proteins in OT-I CTLs. OT-I CTLs were sorted after incubation with OVA-loaded EL-4 cells in the absence or presence of aTregs for 5 h. (h) Flow cytometry analysis of intracellular IP3 receptor expression (left) and total inositol phosphate (IP) production (right) measured in OT-I CTLs (n=3). (i) Maximum intracellular calcium release was measured by Fluo-4 intensity in OT-I CTLs after stimulation with anti-CD3 and anti-CD28 Abs followed by additional ionomycin (1 µM) stimulation at 400 s (OT-I CTL alone, n=19; OT-I CTL+aTreg, n=13). (j) Cells were further analysed by western blot (4-12% SDS–PAGE) for NFAT1 phosphorylation (n=3). (k) OT-I CTLs were cocultured with aTreg in the absence or presence of SB431542 for 16 h. PKH-26 labeled OVA peptide-loaded EL-4 cells were added to the cells for 5 h and apoptotic EL-4 cells were stained for Annexin V (n=3). Throughout, data are the mean±s.e.m. NS: non-significant; P<0.01; P<0.05; P<0.005; by two-tailed Student's t-test.