Figure 1.
Generation of specific anti-human Orai1 monoclonal antibody.
A) Representation of human Orai1 tetra-membrane spanning protein with two extracellular loops of approximately 20 and 40 amino acids, respectively. B) Titration of purified monoclonal anti-Orai1 antibody binding to loop 2 peptide in ELISA assay. C) Ba/F3 cells overexpressing human (h)Orai1, Orai2, or Orai3 or D) Jurkat cells transduced with Orai1-targeting shRNAi or scramble control were incubated with purified anti-Orai1 at the indicated concentrations and detected with fluorophore-conjugated goat anti-mouse IgG. MFI indicates median fluorescence intensity. The experiment in panel B was performed in duplicate and is representative of three individual experiments. Panel C is from single wells from one experiment and panel D is from single wells and is representative of two individual experiments.
Figure 2.
Anti-Orai1 antibody binds to native Orai1 on primary immune cells.
A) Representative histogram demonstrating αOrai1 or isotype control binding to primary immune cells (CD3+CD4+, CD3+CD8+, and CD19+) from the peripheral blood of a healthy individual using 12.5 µg/mL antibody. B) Median Fluorescence Intensity (MFI) of αOrai1 and mIgG1 control binding to memory (IgD−CD27+) and naïve (IgD+CD27−) CD19+ B cells; antibody used at 12.5 µg/mL. C) Binding of αOrai1 and mIgG1 control to naïve (CD45RA+) and memory (CD45RO+) CD3+CD4+ and CD3+CD8+ T cells. D) Binding of aOrai1 to non-lymphocyte NK cells (CD3−CD19−CD14−CD56+), CD86+ and CD86− dendritic cells (CD3−CD19−CD14−CD11c+HLA-DR+), CD14+ monocytes, and granulocytes. All data is representative of at least three independent donors.
Figure 3.
Anti-Orai1 antibody inhibits calcium flux and induces internalization in T cells.
A) Inhibition of thapsigargin-induced calcium flux in Jurkat cells with αOrai1. Data is calculated as percentage of ΔRFU following calcium influx in the absence of antibody addition. B) Representative trace of thaspigargin (TG)-induced calcium influx in Jurkat cells treated with 50 µg/mL αOrai1 or mIgG1 control. C) αOrai1 internalization in purified human CD4+ cells was measured following incubation at 37°C by flow cytometry using 2 µg/mL αOrai1-AF647 and cell surface detection with biotinylated anti-Cy5 followed by streptavidin-BV421. Samples at 4°C were analyzed in parallel as negative controls for internalization. Data is calculated as percentage of total CD4+ cells and is the average of three donors. D) Representative flow cytometry plots of surface bound αOrai1 compared to total internalized and surface αOrai1. Panels A and B are representative of three independent experiments and panels C and D are representative of two experiments.
Figure 4.
Anti-Orai1 antibody inhibits T cell response and proliferation.
A) Proliferation of PBMCs treated with αCD3/αCD28 in the presence of control mIgG1, αOrai1, or cyclosporine A (CsA). Data is calculated as percentage of CFSE-diluted cells in the absence of inhibitor. B) Representative CFSE dilution traces of αCD3/αCD28-treated PBMCs without inhibitor, or with 333 nM CsA or αOrai1 (equivalent to 50 µg/mL). FACS plots are gated on viable CD5+ T cells. C) Effect of treatment with 20.8 nM CsA or αOrai1 (equivalent to 3.1 µg/mL) on IL-2 and IFN-γ production at 16 and 72 h, respectively, following stimulation with αCD3/αCD28. D) Representative CFSE dilution traces of tetanus toxoid-induced proliferation of PBMCs. FACS plots are gated on viable CD3+CD4+ T cells. **P<0.01, ***P<0.001. Panels A–C are representative of three independent experiments (each with at least two donors) and panel D is from a single experiment.
Figure 5.
Orai1 expressing cells are abundant in synovial tissue from patients with rheumatoid arthritis.
Sections of synovial tissue from patients with rheumatoid arthritis or healthy controls were stained for Orai1 by immunohistochemistry with rabbit anti-Orai1 (Sigma), and the number of Orai1-positive immune cells was evaluated by assigning each stained tissue section a semi-quantitative score ranging from 0–4. A) Orai1 immunohistochemistry of rheumatoid arthritis synovial tissue. B) Magnification of A. C) Orai1 immunohistochemistry of normal synovial tissue. D) Histogram showing mean semi-quantitative scores of the number of Orai1-positive cells in synovial tissue from rheumatoid arthritis patients (RA) (n = 24) or normal synovial tissue (n = 11). Error bars, +/− SEM. Scale bars, 100 µm.
Figure 6.
Anti-Orai1 reduces cytokine production by RA synovial fluid cells.
A) Representative forward and side scatter of synovial fluid cells (SFCs) with gating of lymphocytes (lym), monocytes (mono), and neutrophils (neut) and surface staining of Orai1 on CD4+, CD8+, and CD19+ lymphocytes, CD14+ monocytes, and CD66b+ neutrophils from RA synovial fluid. B) IL-2 and C) IFN-γ secretion from αCD3/αCD28 costimulated SFCs following 40 hour-culture in the presence of mIgG1 isotype control, αOrai1, or cyclosporine A (CsA). D) Proliferation of RA patient PBMCs treated with SEB in the presence of control mIgG1, anti-Orai1, CTLA4-Ig, or cyclosporine A (CsA). Data is calculated as percentage of CFSE-diluted cells in the absence of inhibitor. E) Representative CFSE dilution traces of SEB-induced proliferation of RA PBMCs in the presence of 333 nM inhibitor. All panels are representative of two independent experiments.
Figure 7.
Anti-human Orai1 antibody attenuates xenogeneic GvHD in humanized mice.
Groups of humanized NOG mice were treated with 10/kg αOrai1 (N = 11) or mIgG1 isotype control (N = 12) 3 times per week throughout the study. Mice without PBMC transfer were included as controls (N = 3). A) Staining of Orai1 on human CD4+ and CD8+ T cells from spleens of humanized mice following the onset of GvHD. B) Kaplan-Meier curves depict percentage of mice without GvHD defined as <20% weight loss. C) Absolute numbers of human CD4+ and CD8+ T cells in blood were quantified by flow cytometry on indicated days post PBMC injection. D) Human IFN-γ in plasma was measured by ELISA where horizontal dotted lines indicate assay range. E) Paraffin-embedded sections of livers and lungs were immunohistochemically stained with anti-human CD8 and analyzed using automated image analysis software (VIS) to show the CD8 density (% tissue area stained with CD8). Data are individual or mean +/− SEM and representative of two separate experiments. **P<0.01 by Mantel-Cox Log-Rank test compared to PBMCs+mIgG1 (B). *p<0.05, **P<0.01, ***P<0.001 by Mann-Whitney U-test (B-D). In (D), samples above range were given the maximum assay value and included in statistics whereas values below detection were plotted with the value 1, but not included in statistics.