Fig. 5: INPP5E modulates PI(4,5)P₂ environment at the immune synapse.

a Immunostaining was performed in PH-PLCδ-GFP transfected Jurkat T cells in conjugates. Images show in the xy plane (scale bar, 10 μm) or 1.0 μm 3D-reconstructed en face at the T-cell-APC contact site in the xz plane (scale bar, 3 μm). Cells were conjugated for 10 min and stained with an anti-GFP antibody. Phalloidin staining was used to visualize F-actin. Dotted white squares indicate the regions that were selected for 3D reconstruction. b Quantification of PH-PLCδ-GFP clearance at the immune synapse in conjugates. n = 54 conjugates for siCtrl, and n = 74 conjugates for siINPP5E cells. Unpaired t-test. **P < 0.01. c Jurkat cells were transfected with either siCtrl or siINPP5E at day 1, while PH-PLCδ-GFP and LifeAct-TagRFP were transfected at day 3. Cells were analyzed at day 4. Live images of cell conjugates were recorded for 5 min. Arrows indicate localization of PH-PLCδ-GFP at the T-cell-APC contact site. The representative images were shown. 3–5 cells were recorded for each condition. The data were obtained from two independent experiments. Arrows indicate PH-PLCδ at IS sites. d The 3D-reconstructed en face at immune synapse in the yz plane after 5 min conjugation. The dotted white squares indicate the regions that were selected for 3D reconstruction. e Jurkat cells were transfected with expression vectors encoding mCherry and mCherry-PIPKIγ. Immunostaining of CD3ζ-GFP in conjugates of Jurkat T cells and CMAC-labeled APCs, in the presence of SEE. Phalloidin staining was used to visualize F-actin. Upper, mCherry vector transfected cells; lower, mCherry-PIPKIγ transfected cells. Scale bar = 10 μm. f The CD3ζ-GFP recruitment index was quantified. N = 4. n = 71 conjugates for mCherry vector transfected cells, n = 62 conjugates for mCherry-PIPKIγ transfected cells. Error bars indicate mean ± SD. Unpaired t-test. ****P < 0.0001. g Jurkat cells were transfected with either siCtrl or siINPP5E. Immunostaining of PLCγ1 and INPP5E were performed in conjugates of Jurkat T cells and CMAC-labeled SEE-pulsed APCs. Arrows indicate localization of INPP5E. h The PLCγ1 recruitment index was quantified in conjugates. N = 2. n = 37 conjugates for siCtrl, and n = 40 conjugates for siINPP5E. Scale bar = 10 μm. Error bars indicate mean ± SD. Unpaired student T-test. *P < 0.05.