Anti-CD21 antibodies are not significantly internalized, while anti-CD19 antibodies only internalize readily in CD21 or CD21 cells

<p><b>Copyright information:</b></p><p>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"</p><p></p><p>British Journal of Haematology 2007;140(1):46-58.</p><p>Published online 07 Nov 2007</p><p>PMCID:PMC2228374.</p><p>© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd</p> Various B-cell lines were incubated with anti-CD21 (HB135) for 20 h at 37°C in the presence of lysosomal protease inhibitors, and the total antibody distribution detected post fixation and permeabilization with Cy3-conjugated anti-mouse (left panels). Insets show surface binding of anti-CD21 following 1 h incubation on ice. Ramos (A) and DoHH2 (B) cells lack surface expression of CD21 and consequently failed to internalize any antibody, as expected. Anti-CD21 is not significantly internalized in the low CD21-expressing Namalwa (C) or Daudi (D) cells, or even in the higher expressing ARH77 (E) or Raji (F) cells, or in freshly isolated primary human B-cells (G). The same cell lines were incubated with anti-CD19 (B496) antibodies on ice for 1 h (insets in middle panels), or at 37°C for 3 h (middle panels) or 20 h (right panels) with detection as above. The CD21-negative cell lines Ramos (H,O) and DoHH2 (I,P) readily internalized anti-CD19 within 3 h, while the low CD21-expressing Namalwa (J,Q) and Daudi (K,R) cells internalized it less extensively, as judged by the faint plasma membrane staining remaining even after 20 h uptake. The high CD21-expressors, ARH77 and Raji did not detectably internalize anti-CD19 after 3 h (L,M), and after 20 h still had not internalized nearly as much as the CD21-negative cells did in 3 h (S,T). Primary human B-cells did not internalize anti-CD19 within 3 h (N), but did by 20 h (U). Virtually all the cells in each field readily internalized Alexa488-transferrin (with the exception of transferrin-receptor negative primary B-cells), indicating that any lack of antibody uptake was not due to loss of viability (not shown). Gamma levels were adjusted where appropriate. Scale bar = 20 μm

Anti-CD19 is internalized by dynamin-dependent, clathrin-mediated endocytosis and is delivered to lysosomes

<p><b>Copyright information:</b></p><p>Taken from "High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate"</p><p></p><p>British Journal of Haematology 2007;140(1):46-58.</p><p>Published online 07 Nov 2007</p><p>PMCID:PMC2228374.</p><p>© 2007 Genentech, Inc. Journal Compilation © 2007 Blackwell Publishing Ltd</p> (A) Ramos cells were pre-incubated for 30 min at 37°C with the following reagents: dimethyl sulphoxide (DMSO) (1); 1 μmol/l chlorpromazine (Cpmzn) (2), a clathrin-mediated endocytosis inhibitor; 80 μmol/l dynamin inhibitor dynasore, preincubated for 5 min only (3); 2 mmol/l methyl-β-cyclodextrin (MbC) (4) or 5 μg/ml filipin (5), both inhibitors of caveolar and lipid raft endocytosis. Alexa488-anti-CD19 (black bars) or Alexa488-transferrin (grey bars) were then added in the continuous presence of inhibitors for 30 min and surface quenched as in . Results were plotted as a percentage of uptake compared with the DMSO control and represent the average and standard deviation of three independent triplicate experiments. (B–D) Alexa488-anti-CD19 (green channel in B and D) was co-internalized with Alexa647-transferrin (shown in the red channel in C and D) in Ramos cells for 5 min, surface quenched with anti-Alexa488, fixed and imaged. (E–G) Alexa488-anti-CD19 (green channel in E and G) was chased for 3 h in Ramos cells in the presence of lysosomal protease inhibitors prior to fixation and staining with Alexa555-anti-LAMP1 (red channel in F and G). Yellow colour in the merged images in panels D and G indicates colocalization. Gamma levels were adjusted where necessary to better illustrate marker overlap. Arrows indicate examples of co-localized staining. Scale bar is 20 μm in the main panels and 6·7 μm in the 3×-magnified insets of the boxed region indicated in D