Journal: British Journal of Haematology
Article Title: High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate
Figure Lengend Snippet: Quantitation of CD19 and CD21 surface levels and anti-CD19 uptake by flow cytometry confirms the immunofluorescence results. (A) B-cell lines were incubated on ice with 2 μg/ml mouse anti-CD21 (HB135) or mouse anti-CD19 (B496), followed by rat anti-mouse-phycoerythrin and analyzed by flow cytometry to determine surface expression. Results are the average mean fluorescence intensity (MFI) of triplicates ± standard deviation from a representative of three independent experiments (average of five independent experiments shown for the more variable ARH77 cells). Shown in increasing order of CD21 expression are: (1) SuDHL-4, (2) Ramos, (3) DoHH2, (4) Namalwa, (5) Daudi, (6) Ramos-CD21 clone 3, (7) ARH77, (8) Raji, (9) Ramos-CD21 clone 1. (10) Freshly isolated human B-cells have lower fluorescence values for both antigens than expected due to their small size, but their relative ratio of CD21 to CD19 is similar to that of ARH77 and Raji cells. Ramos-CD21 clone 1 expresses CD21 even more highly than Raji, while Ramos-CD21 clone 3 is intermediate between that of ARH77 and Daudi. (B) The rate of internalization of Alexa488-anti-CD19 in Ramos (▪), Ramos-CD21 clone 1 (□), Ramos-CD21 clone 3 (△) and CD21 hi ARH77 (▴) cells was determined by pre-binding to cells then incubating at 37°C (without washing) for the indicated times, washing and fixing either with or without surface fluorescence quenching with anti-Alexa488. Results are the average and standard deviation of two duplicate experiments each normalized to their respective initial surface binding levels after subtraction of background signals.
Article Snippet: Antibodies Unless otherwise indicated, antibodies used were mouse anti-CD19 (clone B496, Biomeda CB-19; Biomeda, Foster City, CA, USA) and mouse anti-CD21 [HB135 (American Type Culture Collection, Manassas, VA, USA), also called THB-5 or HB5], anti-CD20 (2H7), anti-CD22 (RFB4) and anti-CD79b (SN8), all affinity purified at Genentech Inc. (South San Francisco, CA, USA) from hybridoma supernatants.
Techniques: Quantitation Assay, Flow Cytometry, Cytometry, Immunofluorescence, Incubation, Expressing, Fluorescence, Standard Deviation, Isolation, Binding Assay