Journal: British Journal of Haematology

Article Title: High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate

doi: 10.1111/j.1365-2141.2007.06883.x

Figure Lengend Snippet: Anti-CD21 antibodies are not significantly internalized, while anti-CD19 antibodies only internalize readily in CD21 lo or CD21 − cells. 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.

Article Snippet: Conjugates were synthesized as described ( Polson et al , 2007 ), except that the antibodies used were the anti-CD21 clone HB135, anti-CD19 clone B496 and Trastuzumab anti-HER2 (Genentech Inc.).

Techniques: Incubation, Binding Assay, Expressing, Isolation, Clinical Proteomics, Membrane, Staining

Journal: British Journal of Haematology

Article Title: High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate

doi: 10.1111/j.1365-2141.2007.06883.x

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: Conjugates were synthesized as described ( Polson et al , 2007 ), except that the antibodies used were the anti-CD21 clone HB135, anti-CD19 clone B496 and Trastuzumab anti-HER2 (Genentech Inc.).

Techniques: Quantitation Assay, Flow Cytometry, Immunofluorescence, Incubation, Expressing, Fluorescence, Standard Deviation, Isolation, Binding Assay

Journal: Diagnostics

Article Title: Collision Tumor of Angioimmunoblastic T-Cell Lymphoma and Kaposi Sarcoma in an HIV-Negative Elderly Woman: The First Reported Case in Asia

doi: 10.3390/diagnostics15182411

Figure Lengend Snippet: ( A – C ) Scan view images of three lymph nodes show coexistence of Angioimmunoblastic T-cell lymphoma (AILT) and Kaposi sarcoma (KS). ( D – K ) AILT areas show diffuse effacement of lymph node architecture ( D ), polymorphous lymphoid cell infiltration with high endothelial venules ( E ), atypical lymphoid cells with clear cytoplasm ( F ). CD21 staining shows extrafollicular follicular dendritic cells meshworks ( G ). Immunohistochemistry shows the neoplastic clear cells are positive for CD3 ( H ), CD10 ( I ), PD-1 ( J ), and Bcl-6 ( K ). ( L – O ) KS areas show capsular and pericapsular spindle cell proliferation with red blood cell extravasation and slit-like vascular spaces ( L , M ). The neoplastic spindle tumor cells express HHV-8 LANA-1 ( N ) and ERG ( O ).

Article Snippet: After deparaffinization, rehydration, and appropriate antigen retrieval procedures, the sections were incubated with the following primary antibodies: CD3 (Dako, 1:300), CD4 (Dako, 1:200), CD10 (Novocastra, 1:100), CD20 (Dako, 1:200), CD21 (Novocastra, 1:100), BCL6 (Dako, 1:50), CD31 (Dako, 1:300), CD34 (Dako, 1:300), anti-ERG (Roche, prediluted), HHV-8 (Cell Marque, prediluted), and PD-1 (Cell Marque, 1:500).

Techniques: Staining, Immunohistochemistry