Journal: bioRxiv

Article Title: Non-canonical Hedgehog signaling through L-type voltage gated Ca 2+ channels controls CD8 + T cell killing

doi: 10.1101/2021.03.01.433424

Figure Lengend Snippet: ( A ) Naïve CD8 + T cells (left panel) or CTLs (middle panel) were stimulated with plate-bound anti-CD3/CD28 or anti-CD3 antibodies alone for 3 hours in the presence of indicated doses of the cell permeable Ca 2+ chelator BAPTA-AM or carrier control. RNA was extracted for qRT-PCR analysis. Immunoblot analysis of Gli1 and tubulin of CTLs left unstimulated or restimulated with plate-bound anti-CD3 for 3 hours in the presence of BAPTA-AM or carrier control is shown (right panel). Molecular masses are shown in kilodaltons. n=3 independent experiments. ( B ) CTLs were restimulated for 3h with plate-bound anti-CD3 in the presence of 1.25mM BAPTA, 1.25mM EGTA or carrier control before RNA was extracted for qRT-PCR analysis (right panel). n=3 independent experiments. ( C ) Schematic overview of Ca 2+ channels with respective antagonists (red) used in this study. ( D ) CTLs were restimulated with plate-bound anti-CD3 in the presence of the indicated inhibitors or carrier control for 15h before RNA was extracted for qRT-PCR analysis. n=3-4 independent experiments. ( E ) CTLs were restimulated with 10µg/ml cross-linked soluble anti-CD3 in the presence of Nifedipine, 10µM U0126 or carrier control. Cells were subsequently prepared for intracellular flow cytometric analysis. n=2 independent experiments. ( F ) Naïve CD8 + T cells were stimulated with plate-bound anti-CD3ε in the presence of the indicated doses of Nifedipine or carrier control. n=3 independent experiments. ( G, H ) Activated CD8 + T cells were electroporated with RNP complexes at day 2 post stimulation to generate Cav1.3/1.4 -/- (KO) or non-targeting control (NTC) CTLs. CTLs were restimulated on day 8/9 with 10µg/ml cross-linked soluble anti- CD3ε for flow cytometric analysis of pErk ( G ) or restimulated with plate-bound anti-CD3 for qRT-PCR analysis ( H ). n=3 independent experiments. (A,B,D,F,H) Data is normalized to CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Error bars indicate SD. p values were calculated using an unpaired two-tailed Student’s t test (A,B,D,F) or two-way ANOVA (H) and * indicates p<0.05, ** p<0.01, and *** p<0.001.

Article Snippet: Blocking buffer was aspirated and blocking buffer containing primary rabbit anti-Cav1.4 (1:50, Stratech, Cat no. LS-C94032-LSP) antibody was added.

Techniques: Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Non-canonical Hedgehog signaling through L-type voltage gated Ca 2+ channels controls CD8 + T cell killing

doi: 10.1101/2021.03.01.433424

Figure Lengend Snippet: Naïve human CD8 + T cells were isolated from peripheral blood of healthy donors. ( A ) Cells were processed for qRT-PCR analysis of human Cav1.1 ( CACNA1S ), Cav1.2 ( CACNA1C ), Cav1.3 ( CACNA1D ) and Cav1.4 ( CACNA1F ) channel expression. Values were normalized to TBP as a reference gene. ND: Not Detected. Error bars indicate SEM. n=7 donors. ( B ) RNA was extracted before and after stimulation with human T-activator CD3/CD28 beads for 2 days in the presence of 100 µM nifedipine or DMSO (carrier control). Error bars indicate SEM. n=11 donors. ( C ) T cells were stimulated with human T-activator CD3/CD28 beads for 3 days. On d11, CTLs were stained with antibodies against CaV1.4, phalloidin and Hoechst. Scale bar=10 µm. n=8 donors, representative donor shown. ( D ) T cells were stimulated with human T-activator CD3/CD28 beads for 3 days and again restimulated with T-activator CD3/CD28 beads on d10 for 3 days. On d14-d15, CD8 + T cells were co-cultured with P815 target cells at indicated effector to target ratios and subjected to an LDH cytotoxicity assay in the presence of 100µM Nifedipine or DMSO (carrier control). Left panel: Error bars indicate SD, representative donor shown. Right panel: Error bars indicate SEM, n=6 donors. p values were calculated using two-way ANOVA. * indicates p<0.05, and ** p<0.01.

Article Snippet: Blocking buffer was aspirated and blocking buffer containing primary rabbit anti-Cav1.4 (1:50, Stratech, Cat no. LS-C94032-LSP) antibody was added.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Staining, Cell Culture, LDH Cytotoxicity Assay