Figure 6. Downmodulation of CD244 is mediated by endocytosis. A. Schematic summary of tetramer assay.

pHrodo-Red^™produces minimal fluorescence at neutral pH, and fluoresces brightly at low pH. Specific or isotype control antibody was complexed with pHrodo-Red-avidin at a molar ration of 4:1 to form antibody tetramers. Tetramers were allowed to bind to CD8 T cells, and then CD8 T cells were stimulated with PHA or medium-only control. Internalization and transfer of tetramers to an acidic compartment was determined by fluorescent detection of pHrodo-red signal on a flow cytometer. B. pHrodo-red detection in activated CD8 T cells. A cultured CD8 T cell line was stained with anti-CD244 or isotype control tetramers and stimulated with PHA for five hours. Cells were fixed and stained for viability and T cell markers. Plots are gated on live CD8⁺ T cells. C. Internalization of anti-CD244 tetramers induced by simultaneous signals via CD244 and TCR. A cultured CD8⁺ T cell line was loaded with specific or isotype tetramer at the indicated concentrations for 30 minutes, washed and then stimulated with PHA or medium control. Cells were incubated at 37° for five hours, and then stained for FACS, fixed, and analyzed by flow cytometry. Results are representative of two similar experiments. The % pHrodo positive cells across multiple treatments was significantly greater for anti-CD244 tetramer compared to isotype control (Wilcoxan p<0.001).