Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function

doi: 10.4049/jimmunol.1501406

Figure Lengend Snippet: Naïve CD4+ T cells from spleen and LNs of WT and Orai1fl/fl Cd4-Cre mice were stimulated with anti-CD3/CD28 and cultured for 3 days in vitro under Th1, Th17 and iTreg polarizing conditions. (A–C) SOCE was measured in Fura2-AM loaded cells after stimulation with anti-CD3-Biotin and crosslinking with streptavidin followed by readdition of 1 mM Ca2+ to the extracellular medium. (D, E) Frequencies of IFN-γ+, IL-17+ and GM-CSF+ CD4+ T cells measured after stimulation with PMA and ionomycin for 6 h in the presence of Brefeldin A. Bar graphs represent the average ± SEM from 4 independent experiments with one mouse per group. (F, G) Relative mRNA expression of Tbx21 and Rorc in Th1 and Th17 cells cultured in vitro (left) and in lymphocytes isolated from the spinal cord of WT and Orai1fl/fl Cd4-Cre mice on day 27 after immunization with MOG35-55 peptide in CFA (right). Tbx21 and Rorc mRNA levels were analyzed by real-time PCR (and normalized to Hprt1 (left panels) or CD4 in right panels). Bar graphs represent the average ± SEM of 4–6 mice per group. (H, I) Flow cytometric analysis of T-bet and RORγt in Th1 and Th17 polarized cells. WT control in the left panel are Th17 polarized cells and WT control in the right panel are Th1 polarized cells. Bar graphs represent the average ± SEM from 3 mice per group. (J) Frequencies of Foxp3+ CD4+ iTreg cells after 3 days in vitro without restimulation. Bar graphs represent the average ± SEM from 4 independent experiments with one mouse per group. (K) ORAI1 is not required for iTreg function. WT or Orai1fl/fl Cd4-Cre iTreg cells that had been differentiated for 3 days in vitro were coincubated at different ratios with CFSE-labeled, anti-CD3-stimulated CD4+ T cells and splenocytes from WT mice. Proliferation of CD4+ T cells was assessed after 3 days in culture by CFSE dilution and flow cytometry. Shown is one representative experiment done in triplicates of two independent experiments. No statistically significant difference of proliferation was detected between T cells incubated with WT and Orai1-deficient Treg cells at different ratios by one-way ANOVA. Statistical analysis in D-J was performed using an unpaired Student’s t test. ** p < 0.01, **** p < 0.0001.

Article Snippet: For stimulation by TCR crosslinking, T cells were incubated with 1 μg/ml Biotin-conjugated anti-CD3ε antibody (145-2C11, BD Biosciences) at the time of Fura-2 loading and stimulated by addition of 1 μg/ml Streptavidin (Invitrogen), followed by addition of 1 mM Ca 2+ Ringer solution and in some experiments 0.3 μM ionomycin.

Techniques: Cell Culture, In Vitro, Expressing, Isolation, Real-time Polymerase Chain Reaction, Control, Labeling, Flow Cytometry, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function

doi: 10.4049/jimmunol.1501406

Figure Lengend Snippet: CD4+ T cells isolated from the spleen and LNs of WT were stimulated with anti-CD3/CD28 for 3 days in vitro under Th1, Th17 and iTreg polarizing conditions. The CRAC channel inhibitor AMG1 (100 nM) or vehicle (DMSO) was added on day 0 and was present throughout T cell differentiation (day 0–3) (A–C) or added on day 3 immediately before Ca2+ measurements (D–F, left panels) or restimulation to measure cytokine production (D–F, right panels). For Ca2+ measurements (left panels), CD4+ T cells were loaded with Fura2-AM, incubated with Biotin-labeled anti-CD3 and activated by CD3 crosslinking with streptavidin in Ca2+ free extracellular buffer. To induce SOCE, extracellular [Ca2+] was raised to 1 mM. For cytokine production, CD4+ T cells were stimulated with PMA and ionomycin for 6 h. For analysis of Foxp3 expression, CD4+ T cells were left unstimulated. Data represent the average ± SEM of 3–8 mice per group. Statistical analysis was performed using an unpaired Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001.

Article Snippet: For stimulation by TCR crosslinking, T cells were incubated with 1 μg/ml Biotin-conjugated anti-CD3ε antibody (145-2C11, BD Biosciences) at the time of Fura-2 loading and stimulated by addition of 1 μg/ml Streptavidin (Invitrogen), followed by addition of 1 mM Ca 2+ Ringer solution and in some experiments 0.3 μM ionomycin.

Techniques: Isolation, In Vitro, Cell Differentiation, Incubation, Labeling, Expressing