orai1  (Alomone Labs)

Journal: Frontiers in Pharmacology

Article Title: A Compartmentalized Reduction in Membrane-Proximal Calmodulin Reduces the Immune Surveillance Capabilities of CD8 + T Cells in Head and Neck Cancer

doi: 10.3389/fphar.2020.00143

Figure Lengend Snippet: Decreased membrane CaM in HNSCC T cells. (A) Representative confocal images of HD and HNSCC T cells stained for CD8 (green; no permeabilization) and CaM (magenta, after permeabilization). Scale bars indicate 10 μm. Representative individual cells highlighted (box) in the merged images are magnified in the extreme right panels. Co-localization is indicated by white color. (B, C) Fluorescence intensities of CaM in the membrane (B) and cytoplasm (C) of HD and HNSCC T cells were measured as described in Materials and Methods . Data represented by a box and whisker plot. The horizontal line represents median value of CaM fluorescence intensities for each group, while the first and third quartiles are represented in the top and bottom boxes respectively. (D) Ratio of CaM fluorescence at the cell membrane and cytoplasm of HD and HNSCC T cells. Bars represent mean ± SEM (E) Representative confocal images of HD and HNSCC T cells stained for KCa3.1 (green). Scale bars indicate 10 μm. (F) Fluorescence intensities of KCa3.1 in the membranes of HD and HNSCC T cells. Bars represent mean ± SEM of 117 cells from 3 HD and 61 cells from 3 HNSCC patients. Shown in (B–D) are values for 96 cells from 3 HD and 110 cells from 3 HNSCC patients. Data in (B–D, F) were analyzed by Mann Whitney rank sum test.

Article Snippet: Activated CD8 + T cells from HDs and HNSCC patients were seeded on poly-L-lysine-coated coverslips, fixed with 4% paraformaldehyde, permeabilized with PBS + 0.2% Triton X-100, blocked with PLA blocking solution (Millipore Sigma) for 1 h, and stained with mouse anti-human KCa3.1 (Clone 6C1, Alomone labs) and rabbit anti-human CaM antibodies (clone SJ16-09, Novus Biologicals) diluted in antibody dilution buffer (Millipore Sigma) for 3 h at room temperature.

Techniques: Staining, Fluorescence, Whisker Assay, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: A Compartmentalized Reduction in Membrane-Proximal Calmodulin Reduces the Immune Surveillance Capabilities of CD8 + T Cells in Head and Neck Cancer

doi: 10.3389/fphar.2020.00143

Figure Lengend Snippet: Reduced association of KCa3.1 with CaM in HNSCC T cells (A) Linescan analysis of KCa3.1 and CaM association at the cell membrane. (Left) Magnified representative confocal images of HD and HNSCC T cells stained for KCa3.1 (green) and CaM (magenta) from the merged images in Figure S4 (box). Lines were drawn across the cells for linescan analysis, and representative lines drawn for a linescan are shown. (Right) Line scan analyses were generated using ImageJ software and are plotted as KCa3.1 (green line) and CaM (magenta line) fluorescence intensity (y-axis) vs. the length of the lines drawn across the merged cells (distance from the starting point of the line, μm, x-axis). Shown here are linescan graphs from three representative HD and HNSCC T cells, wherein the outmost KCa3.1 (green) peaks correspond to the cell membrane. Correspondence of the peaks for green (KCa3.1) and magenta (CaM) lines is indicative of their colocalization. (B) Comparison of KCa3.1 membrane expression in HD and HNSCC T cells. KCa.31 fluorescence intensities were measured at the cell surface (points corresponding to KCa3.1 fluorescence peaks on the cell membrane) in linescan plots (A) . The data were normalized to mean KCa3.1 fluorescence intensity measured in HD T cells. (C) Comparison of CaM membrane-associated expression in HD and HNSCC T cells. CaM fluorescence intensities were measured at the cell surface (points corresponding to KCa3.1 fluorescence peaks on the cell membrane) in linescan plots (A) . The data were normalized to mean KCa3.1 fluorescence intensity measured in HD T cells, which represents the proportion of CaM associated with membrane KCa3.1. For (B, C) , bars represent mean ± SEM for data from 108 points (corresponding to KCa3.1 fluorescence peaks in the linescans) analyzed in 18 cells from 3 HD, and 120 points analyzed in 20 cells from 3 HNSCC patients. (D) Interaction between KCa3.1 and CaM in cell membranes of HD and HNSCC T cells. Confocal images of representative cells from PLA experiments performed in HD (top) and HNSCC (bottom). Cells were incubated with rabbit anti-human CaM and mouse anti-human KCa3.1 antibodies, after which PLA probe–ligated secondary antibodies were then added and PLA was performed as described in Materials and Methods . Positive PLA signals showing single protein interactions between KCa3.1 and CaM are visualized as magenta fluorescent dots. Cell membranes are labeled with CD8 antibodies (green) and nuclei are labeled with DAPI (blue). Scale bar, 10 μm. (E) 3D views of the PLA dots (red) in relation to the nucleus (DAPI, cyan) and membrane (CD8, green) in a representative HD (left) and HNSCC (right) T cell. (F) Average PLA signals. PLA signals in the cell membranes of in HD and HNSCC T cells were quantified as number of dots per cell membrane and are represented as a box plot. The dots per cell membrane are reported as median (horizontal line), first (top box), and third quartiles (bottom box). Shown are values for 185 cells from 3 HD and 214 cells from 4 HNSCC patients. For (B, C) , significance was determined by Mann Whitney rank sum test, while the significance for (F) was determined by Student's t-test.

Article Snippet: Activated CD8 + T cells from HDs and HNSCC patients were seeded on poly-L-lysine-coated coverslips, fixed with 4% paraformaldehyde, permeabilized with PBS + 0.2% Triton X-100, blocked with PLA blocking solution (Millipore Sigma) for 1 h, and stained with mouse anti-human KCa3.1 (Clone 6C1, Alomone labs) and rabbit anti-human CaM antibodies (clone SJ16-09, Novus Biologicals) diluted in antibody dilution buffer (Millipore Sigma) for 3 h at room temperature.

Techniques: Staining, Generated, Software, Fluorescence, Expressing, Incubation, Labeling, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: A Compartmentalized Reduction in Membrane-Proximal Calmodulin Reduces the Immune Surveillance Capabilities of CD8 + T Cells in Head and Neck Cancer

doi: 10.3389/fphar.2020.00143

Figure Lengend Snippet: Increased KCa3.1 activity in HNSCC T cells by intracellular delivery of CaM. (A) Representative KCa3.1 currents recorded in whole-cell voltage-clamp configuration in activated T cells from a HNSCC patient and a HD in the presence or absence of 50 μM CAM. The KCa3.1 currents are normalized for the maximum current at +50 mV to ease comparison of the KCa3.1 conductance at hyperpolarizing voltages. (B) KCa3.1 conductance, G (Panel C ) and Kv1.3 peak currents measured in the presence or absence of 50 μM CaM in T cells from HNSCC patients (n = 20 cells without CaM and n = 21 cells with CaM from 4 patients) and HD (n = 20 cells from three individuals). (D) KCa3.1 G measured in the presence 1 μM, 3 μM, and 10 μM free Ca 2+ in the patch pipette in activated HNSCC T cells (n = 18 cells for each conditions from three patients). The values in panels B, C, and D are presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third quartiles (bottom box). The KCa3.1 G or Kv1.3 peak currents were measured in at least five cells for each condition in an individual. Data in (B, C) were analyzed by two-way ANOVA [ (B) : p < 0.001 for HD vs HNSCC and p = 0.046 for + CaM vs. –CaM; (C) : p = 0.359 for HD vs. HNSCC and p = 0.673 for + CaM vs. –CaM], while data in (D) were analyzed by one-way repeated measures ANOVA (p = 0.796).

Article Snippet: Activated CD8 + T cells from HDs and HNSCC patients were seeded on poly-L-lysine-coated coverslips, fixed with 4% paraformaldehyde, permeabilized with PBS + 0.2% Triton X-100, blocked with PLA blocking solution (Millipore Sigma) for 1 h, and stained with mouse anti-human KCa3.1 (Clone 6C1, Alomone labs) and rabbit anti-human CaM antibodies (clone SJ16-09, Novus Biologicals) diluted in antibody dilution buffer (Millipore Sigma) for 3 h at room temperature.

Techniques: Activity Assay, Transferring, Whisker Assay

Journal: Frontiers in Pharmacology

Article Title: A Compartmentalized Reduction in Membrane-Proximal Calmodulin Reduces the Immune Surveillance Capabilities of CD8 + T Cells in Head and Neck Cancer

doi: 10.3389/fphar.2020.00143

Figure Lengend Snippet: Reduction of KCa3.1 activity and increased adenosine sensitivity in HD T cells by CALM1 knockdown. ( A , left) Representative flow cytometry overlay histogram showing CaM expression in activated T cells from one HD transfected with scr-RNA and si CALM1 . (Right) gMFI for CAM expression in T cells from nine HD transfected with scr-RNA and si CALM1 . Transfected cells were identified by positive staining for GFP. (B, C) KCa3.1 conductance (B) and Kv1.3 peak currents (C) in T cells transfected with either si CALM1 (n = 10 cells) or scr-RNA (n = 10 cells) from two HDs. Transfected cells were identified by positive staining for GFP. Horizontal red line represents mean values for each group. (D) Trajectories of T cells co-transfected with GFP and either scr-RNA or si CALM1 migrating along either a CXCL12 gradient (green triangles) or a combination gradient of CXCL12 and adenosine (blue triangles) in a representative HD. Trajectories of GFP positive cells (indicating successful transfection) are shown for each condition. The starting point for each trajectory is artificially set to the same origin. The red triangles represent Y-COM. (E) Y-COM values for cells migrating along either a CXCL12 gradient or a combination gradient of CXCL12 with adenosine in HD T cells transfected with scr-RNA (n = 8 donors) and si CALM1 (n = 8 donors). (F) (Left) Representative flow cytometry plots gated on CD3 + T cells transfected with either scr-RNA or si CALM1 showing IFNγ production after stimulation. Live cell population was identified by exclusion from Zombie Aqua live/dead stain and gated on CD3 + population and transfected cells were identified by positive staining for GFP. IFNγ + T cell populations were selected by drawing rectangle gates. The numbers next to the gates are the percentages of IFNγ + CD3 + T GFP + cells. (Right) Quantification of IFNγ induction in scr-RNA or siCALM1 transfected CD3 + T cells post 48 h activation in three HDs. Bars represent mean ± SEM. All data were analyzed by paired Student's t-test.

Article Snippet: Activated CD8 + T cells from HDs and HNSCC patients were seeded on poly-L-lysine-coated coverslips, fixed with 4% paraformaldehyde, permeabilized with PBS + 0.2% Triton X-100, blocked with PLA blocking solution (Millipore Sigma) for 1 h, and stained with mouse anti-human KCa3.1 (Clone 6C1, Alomone labs) and rabbit anti-human CaM antibodies (clone SJ16-09, Novus Biologicals) diluted in antibody dilution buffer (Millipore Sigma) for 3 h at room temperature.

Techniques: Activity Assay, Flow Cytometry, Expressing, Transfection, Staining, Activation Assay