Fig 2: mRNA expression in EL4 cells stimulated with or without anti-CD3/CD28 antibodies or anti-CD3/CD28/OCILRP2 antibodies.RNA from un-stimulated or CD3/CD28 antibody- or CD3/CD28/OCILRP2 antibody-stimulated EL4 cells was isolated using the RNeasy kit (Qiagen). cDNA of IL-2, NF-κB, NFAT, MAPK3, and MAPK8 or the control household gene β-actin was amplified using SYBR Premix Ex Taq (RRO41A, TaKaRa, Shiga, Japan), and expression was monitored using an Rotor-gene 6000 real-time platform (Corbett Research, Australia). Ct values were normalized for the expression of the β-actin gene. *P<0.05.

Fig 3: OCILRP2 co-stimulation affects EL4 cell viability.Six-well flat-bottom microtiter plates were pre-coated with anti-CD3/CD28 antibodies or anti-CD3/CD28/OCILRP2 antibodies (a) or anti-CD3/CD28/IL-2 antibodies (b) overnight at 4°C. Cells were transfected with pEGFP-siOCILRP2 plasmid and then stimulated with the anti-CD3/CD28 antibodies (a). Then, 100 µL of the cell suspension (5,000 cells/well) was dispensed into each well of a 96-well plate. After incubation for 24, 48, and 72 h (37°C), 10 µL of CCK-8 solution was added to each well, and the cells were incubated for 1 h (37°C). The absorbance was measured at 450 nm using a spectrophotometer (Beckman Coulter DU 640, USA). *P<0.05.

Fig 4: Membrane co-localization of OCILRP2 and DAP12 under anti-CD3/CD28 mAb treatment in EL4 T cells.EL4 T cells pre-transfected with pEGFP-siOCILRP2 or pre-coated with the OCILRP2 Ab were cultured overnight in the presence or absence of an anti-CD3/CD28 mAb and then stained for OCILRP2 (green) and nuclear stained with DAPI (blue) and DAP12 (red) to study OCILRP2 and DAP12 protein expression and localization. Unstimulated EL4 T cells exhibited OCILRP2 protein expression in the cytoplasm (upper panels). In contrast, CD3/CD28-activated EL4 T cells showed intracellular and membrane OCILRP2. OCILRP2/DAP12 co-localization appears in yellow. Each picture is representative of the vast majority of the observed cells on the slides.

Fig 5: Membrane re-localization of OCILRP2 via treatment with an anti-CD3/CD28 mAb.EL4 cells were incubated with plate-bound CD3/CD28 antibodies, isotype-matched mIgGs, or PMA/ionomycin for 48 h. The cells (1×106 cells) were then incubated with an anti-OCILRP2 antagonist antibody for 30 min on ice, followed by staining with an FITC-labeled second antibody. The cells were analyzed with Cellquest software using a FACS Calibur flow cytometer.