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 degrees 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 degrees C for 1 hr prior to APE exposure but not when incubation was carried out at 4 degrees 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 degrees C followed by reaction with APE. However, they failed to react with APE when the 1-hr incubation took place at 37 degrees 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 degrees 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 Lym-1 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.