Internalization and shedding of Lym-1 monoclonal antibody following interaction with surface antigens of a cultured human B cell lymphoma

Modulation of surface antigens of Raji Burkitt's lymphoma cells by monoclonal antibody Lym-1 was investigated by flow cytometry and radioimmunoassay (RIA). Raji cells, treated with antibody conjugated with FITC, became bright green as determined by FACS and the FITC-labeled Lym-1 remained associated with target cells for up to 48 hr after incubation at either 4 or 37 °C. Lym-1 antibody linked with biotin also bound to Raji cells and rendered these cells highly reactive with avidin-phycoerythrin (APE). However, the APE fluorescence intensity measured by FACS decreased substantially when Raji cells were cultured at 37 °C for 1 hr prior to APE exposure but not when incubation was carried out at 4 °C, indicating a disappearance of antibody from the surface of the metabolically active cells. This process was time dependent with a total loss of surface-bound biotinylated antibody occurring over a period of approximately 2 hr. Raji cells exposed to both fluoresceinated and biotinylated Lym-1 in a double labeling experiment became positive to both reagents. The flow cytometric profile was not altered when these cells were incubated for 1 hr at 4 °C followed by reaction with APE. However, they failed to react with APE when the 1-hr incubation took place at 37 °C despite the fact that they remained FITC positive, suggesting that the antibody with its fluorescent label had entered the cells. Utilizing ¹³¹I-labeled Lym-1 it was determined that approximately 50% of initially bound antibody had dissociated from the cells within the first 2 hr of incubation at 37 °C, although the remainder persisted with targets for up to 48 hr. The HPLC protein profile indicated that the radioactivity found in the culture supernatants and cytoplasm was associated with whole antibody, degradation products, and Ig complexes with antigen. Therefore, the present findings suggest that Lym1 Ig molecules react with cell surface antigens and are rapidly internalized and shed, resulting in the disappearance of antibody from the surface membrane of Raji cells.