Phospho-CD3 zeta (Tyr142) Antibody - #AF5425

Fig. 7 CD45 on tumor cells attenuates TCR signaling. a Immunoblot analysis for TCR signaling of Jurkat cells after activation with OKT3 (1 μg/mL) and cocultured with DLD1 cells overexpressing CD45 isoforms for 5 min. b Ca2+ influx of Jurkat cells after OKT3 (5 μg/mL) stimulation and cocultured with DLD1 cells with or without CD45 overexpression. c Immunofluorescence images of Jurkat cells after activation with OKT3 (1 μg/mL) and cocultured with DLD1 cells with or without CD45 overexpression for 5 min. Pearson’s correlation coefficient characterizes the colocalization of CD3ε (green) and CD45 (purple), each point represents one cell. Scale bar = 5 μm. d Immunoblot analysis for HLA-ABC expression of Caco2 cells overexpressing different CD45 isoforms. e Flow cytometric analysis of HLA-A2 expression on Caco2 cells. f Flow cytometric isolation of HLA-A2+ CD3+ T cells from PBMCs of healthy donors. g Immunoblot analysis for TCR signaling of HLA-A2+ CD3+ T cells after activation with OKT3 (1 μg/mL) and cocultured with Caco2 cells with or without CD45 overexpression within 5 min. h Immunoblot analysis for NY-ESO-1 expression in Caco2 cells after the indicated manipulation. i Immunoblot analysis for TCR signaling of Jurkat-TCR cells after activation with OKT3 (1 μg/mL) and cocultured with Caco2-NYESO1 cells with or without CD45 overexpression within 5 min. j Ca2+ influx of Jurkat-TCR cells after OKT3 (5 μg/mL) stimulation in the presence of Caco2-NYESO1 cells with or without overexpression of CD45 isoforms. k IL-2 secretion of Jurkat-TCR cells after cocultured with Caco2-NYESO1 cells with or without CD45 overexpression for 16 h. l Flow cytometric analysis of CD69 expression on Jurkat-TCR cells when cocultured with Caco2-NYESO1 cells with or without CD45 overexpression for 16 h. m, n CD45 exclusion from the immune synapse (IS) on Jurkat-TCR cells or CD8+ T-TCR cells in contact with Caco2-NYESO1 cells with or without CD45 overexpression after 5 min of activation with OKT3 (1 μg/mL), fixed, stained with anti-CD45 and anti-flag antibody. The exclusion percentage = (1-ICD45 in IS zone/ICD45 out IS zone) × 100%. I means intensity. Data are presented as median with 95% CI. Scale bar = 10 μm. o Schematic diagram showing the experimental setup for the investigation of the intercellular CD45-CD45 homophilic interactions. The co-immunoprecipitation data below illustrates the homophilic CD45-CD45 interactions in the cell model. p Immunofluorescence images showing the intercellular CD45-CD45 homophilic interactions detected by proximity ligation assay. Scale bar = 10 μm. q Schematic diagram showing the experimental setup for the investigation of the intercellular CD45-CD45 homophilic interactions. The co-immunoprecipitation data below illustrates the homophilic CD45-CD45 interactions between Caco2 cells overexpressing HA-CD45 and Jurkat-sgCD45-CD45GFP cells. r Immunofluorescence images showing the intercellular CD45-CD45 homophilic interactions detected by proximity ligation assay. Scale bar = 10 μm. s Predicted crystal structure of CD45-CD45 interactions using ZDOCK. t The proximity ligation assay and (u) the co-immunoprecipitation data showing the homophilic CD45-CD45 interactions between HEK293T-HA-CD45 and HEK293T-MYC-CD45 cells with or without CD45 truncation of amino sites 430–436. Scale bar = 10 μm. v Schematic diagram showing the regulation of the TCR-pMHC complex and the potential immuno-modulatory role of CD45-CD45 dimerization between tumor cells and T cells. Unless otherwise specified, all bar graph data are presented as means ± SEM. *P