Introduction: Rituximab (Rtx) is an anti-CD20 monoclonal antibody that is clinically active in B-cell chronic lymphocytic leukemia (B-CLL). In vitro and in vivo data indicate that Rtx mediates its biologic effect through multiple mechanisms including apoptosis, complement dependent cytotoxicity (CDC), and antibody dependent cellular cytotoxicity (ADCC).

Objetive: To study in vitro sensitivity to apoptosis by Rtx of isolated cells obtained from B-CLL patients Binet stage A, in the presence of complement from different sources and correlate with CD20 and CD59 molecules expression.

Methods: PBMCs were isolated from peripheral blood samples by centrifugation on a Ficoll/Hypaque gradient and cryopreserved in liquid nitrogen. Cells from 21 patients were cultured at 1X106 cells/ml in RPMI 1640 culture medium supplemented with 20% AB serum or 10% heat inactivated fetal calf serum (FCSi) with increasing quantities of rabbit complement (RC) added as a source of complement. Apoptosis was determined by Annexin V-FITC Apoptosis Detection Kit. The absolute number of CD20 and CD59 molecules pretreatment was measured using DAKO QIFIKIT (DAKO Denmark). Comparison of apoptosis and antigen expression between models of culture was performed by the t Student test and U-Mann Whitney respectively.

Results: Incubation of cells with AB alone, FCSi with rabbit complement (RC) or Rtx alone did not induce cell death. A Rtx dose-response study was first performed. Rituximab was added at 1ug/ml, 10ug/ml, 50ug/ml and increasing quantities of RC (10, 30, 60 and 120ul/well). The effect was doses dependent for both Rtx and RC. A discriminant cytotoxic effect was observed with 10 μg/mL Rtx in the presence of 60 ul RC. Cells treated with Rtx 100ug/ml and AB serum for 24 hours caused apoptosis in 52% of patients (11/21 with median of 7%)(Model A). However, when cells from these patients were treated with Rtx, FCSi and RC, at much lower concentration, 10ug/ml and 60ul RC, cell death was observed in 69% of patients (13/21 with median of 37%) (Model B) p<0.0001. In order to study the differences between models of culture utilized, we correlated apoptosis results with the expression of the CD20 and CD59 antigens. These results are shown in Table 1.

Conclusions: Rituximab alone was unable to induce a cytotoxic effect on B cells. The addition of a source of complement is necessary to obtain such an effect. This effect was highest when cells were treated with RC and the citotoxicity was doses dependent. Rituximab-induced cell death was significantly associate with high CD20 expression, and also correlate with the CD59/CD20 ratio independently of complement source used.

Correlation between Model A and B, with CD20 and CD59 expression

Sensitive ANO Sensitive Ap
Model A: Rtx 100ug/ml and 20% AB serum; Model B: Rtx 10ug/ml, FCSi and RC; * x 10³molecules/cell 
CD20 expression* 36,4 (17,7–89,9) 18,7 (10,7–28,5) <0.0001 
CD59 expression* 15,6 (10,4–31,6) 16,4 (13,7–24,6) 0.310 
Ratio CD59/CD20 0.42 (0.20–1.31) 0.94 (0.57–1.40) <0.0001 
 Sensitive B NO Sensitive B p 
CD20 expression* 28,0 (18,1–89,9) 18,8 (14,4–28,0) <0.0001 
CD59 expression* 16,7 (10,4 –31,6) 23,3 (16,0–26,7) 0.018 
Ratio CD59/CD20 0.55 (0.20–0.95) 1.18 (0.79–1.44) 0.002 
Sensitive ANO Sensitive Ap
Model A: Rtx 100ug/ml and 20% AB serum; Model B: Rtx 10ug/ml, FCSi and RC; * x 10³molecules/cell 
CD20 expression* 36,4 (17,7–89,9) 18,7 (10,7–28,5) <0.0001 
CD59 expression* 15,6 (10,4–31,6) 16,4 (13,7–24,6) 0.310 
Ratio CD59/CD20 0.42 (0.20–1.31) 0.94 (0.57–1.40) <0.0001 
 Sensitive B NO Sensitive B p 
CD20 expression* 28,0 (18,1–89,9) 18,8 (14,4–28,0) <0.0001 
CD59 expression* 16,7 (10,4 –31,6) 23,3 (16,0–26,7) 0.018 
Ratio CD59/CD20 0.55 (0.20–0.95) 1.18 (0.79–1.44) 0.002 
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Topics:
apoptosis, complement system proteins, culture media, rituximab, cd20 antigens, antigens, cd59, molecule, annexins, antibodies, antigens

Work supported by FIS PI 020889

Author notes

Corresponding author

2005, The American Society of Hematology
2004
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