Fig 2: Optical control of intracellular Ca2+ signal in CD8+ T cells overcomes immunosuppression.(a) 2 × 105 B16F10 cells in 10 µl PBS were intradermally injected into the ear and flank. Expression of CD107a on Pmel-1 CD8+ T cells in B16 tumours in the ear with (optical LED+light) or without (optical LED+dark) light stimulation of tumours in the ear (light, n=5; dark, n=5). (b) Size of B16 tumours both in the ear and flank treated with adoptive transfer of CatCh-expressing Pmel-1 CD8+ T cells with (optical LED+light) or without (optical LED+dark) light stimulation of the ear (light, n=5; dark, n=5). (c) Expression of CD69 on OT-I CD8+ T cells after re-stimulation with N4 peptide (1 µg ml-1) loaded irradiated splenocytes in the absence or presence of CGS21680 (10 µM, n=4). (d) Expression of CD69 on OT-I CD8+ T cells after re-stimulation with N4 peptide (1 µg ml-1) loaded irradiated splenocytes in the absence or presence of PGE2 (5 µM, n=4). (e) OT-I CD8+ T cells were incubated with PBS or sFasL (100 ng ml-1) for 16 h. Apoptotic OT-I CD8+ T cells were stained for Annexin V (n=4). (f) Killing of N4 peptide-loaded EL-4 cells after light stimulation of CatCh- or GFP-expressing OT-I CD8+ T cells in the absence or presence of MDSC (1:1) (n=6).

Fig 3: RIPK3 and/or Casp-1/11 deficiencies reduce antigen-specific CD8+ T cells degranulation and intracellular cytokine production in response to LM-OVA. (A) Representative images of frequency of LAMP-1-positive OVA-specific CD8+ T cells for LM-OVA-infected and non-infected (control) groups and the (B) frequency variation of LAMP-1+-CD8+ T cells observed on the LM-OVA-infected group. Immediately, representative image of intracellular H2-Kb-SIINFEKL-specific (C) IFN-? and (E) TNF-a-producing CD8+ T cell expression on control and infected groups. The frequency of these cell populations, H2-Kb-SIINFEKL-specific (D) IFN-? and (F) TNF-a-producing CD8+ T cells, after 7 days of infection with LM-OVA was schematized. Frequencies described in (B,D,F) bar graphs were representative of experiments done in triplicate, and the data depict mean ± SEM of five individual mice per group. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni post-tests. ***p < 0.001.

Fig 4: Optical control of intracellular Ca2+ signal in CD8+ T cells.(a) Representative fluorescence images of Fluo-4/AM-loaded HEK293 cells during light activation. Scale bar, 100 µm. Intensity traces of mock-transfected cells (Con; n=16) and cells transiently transfected with CatCh (n=20). (b) Western blot analysis of NFAT1 phosphorylation in CatCh-expressing OT-I CD8+ T cells after light stimulation for the indicated time. (c,d) IFN-? secretion and cytotoxicity of CatCh-expressing OT-I CD8+ T cells in the presence of G4 peptide-loaded EL-4 cells with or without light stimulation (n=3). (e) Killing of N4 peptide-loaded EL-4 cells after light stimulation of CatCh- or GFP-expressing OT-I CD8+ T cells in the presence or absence of aTregs (n=3). (f) Expression of perforin, granzyme B and CD107a on WT or Orai1-cKO CD8+ T cells after incubation with N4 peptide-loaded EL-4 cells at a 1:1 ratio for 6 h (n=3). (g) Killing of N4 peptide loaded EL-4 cells by WT or Orai1-cKO CD8+ T cells (n=6). (h) Killing of N4 peptide loaded EL-4 cells by CatCh-expressing WT or Orai1-cKO CD8+ T cells with light stimulation (463 nm) (n=3). Throughout, data are the mean±s.e.m. and were analysed by two-tailed Student's t-test.

Fig 5: 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.