1. Conjugation of Blocked Ricin to an Anti-CD19 Monoclonal Antibody Increases Antibody-Induced Cell Calcium Mobilization and CD19 Internalization
by Anne-Christine Goulet, Victor S. Goldmacher, John M. Lambert, Chantal Baron, Denis-Claude Roy, and Edouard Kouassi
Blood
Volume 90(6):
September 15, 1997
©1997 by American Society of Hematology

2. Time-dependent and cell-specific effects of anti-B4–bR and anti-B4 on intracellular calcium mobilization.
Time-dependent and cell-specific effects of anti-B4–bR and anti-B4 on intracellular calcium mobilization. Namalwa, Daudi, and Molt-4 cells were loaded with the Ca2+ indicator Fluo-3-AM and then stimulated with a concentration of 5 nmol/L of unconjugated anti-B4 MoAb (anti-CD19), or anti-B4–bR (anti-CD19-bR) followed by GAM. In some experiments, Molt-4 cells were stimulated with ionomycin (3 μmol/L). Agents were added at 125 seconds as indicated by the arrows, and fluorescence was measured every second with no interruption of sample flow during the data collection period (from 0 to 512 seconds). Data are expressed as dot plots of fluorescence versus time. Traces are representative of eight, five, and two separate experiments with Namalwa, Daudi, and Molt-4 cells, respectively.
Anne-Christine Goulet et al. Blood 1997;90:
©1997 by American Society of Hematology

3. Lack of effect of anti-CD20–bR (A) and anti-CD–38-bR (B) on calcium mobilization in Namalwa and Daudi cells.
Lack of effect of anti-CD20–bR (A) and anti-CD–38-bR (B) on calcium mobilization in Namalwa and Daudi cells. The experimental conditions are similar to those described in Fig 1.
Anne-Christine Goulet et al. Blood 1997;90:
©1997 by American Society of Hematology

4. Effect of EGTA and tyrphostin on [Ca2+]i mobilization induced by anti-B4–bR in Namalwa cells.
Effect of EGTA and tyrphostin on [Ca2+]i mobilization induced by anti-B4–bR in Namalwa cells. (A) Anti-B4–bR induces both mobilization of intracellular Ca2+ and extracellular Ca2+ influx. Namalwa cells were pretreated with EGTA (0.5 mmol/L; Δ) at 60 seconds to chelate extracellular Ca2+ and were stimulated with the IT anti-B4–bR (5 nmol/L) at 125 seconds. CaCl2 (0.5 mmol/L) was then added at 662 seconds to restore extracellular Ca2+. Response of control cells without pretreatment with EGTA, but with stimulation with IT and addition of Ca2+ are included (•). Data are expressed as HMFI versus time. (B) Dose-dependent effect of tyrphostin on [Ca2+]i response induced by anti-B4–bR. Namalwa cells were pretreated for 18 hours at 37°C with increasing concentrations of tyrphostin (0 μmol/L; •); (10 μmol/L; □); (30 μmol/L; ▴); (50 μmol/L; ▵), (100 μmol/L; ▪), and (200 μmol/L; ○). Cells were then loaded with Fluo3-AM and analyzed by flow cytometry for IT-induced calcium mobilization. Data are expressed as HMFI versus time, and traces are representative of three experiments.
Anne-Christine Goulet et al. Blood 1997;90:
©1997 by American Society of Hematology

5. Dose-dependent effects of dimeric and monomeric fractions of anti-B4–bR on calcium mobilization.
Dose-dependent effects of dimeric and monomeric fractions of anti-B4–bR on calcium mobilization. Dimers and monomers of IT were purified by gel filtration and tested for calcium mobilization in Namalwa cells. Data are expressed as the peak values of percent increase in fluorescence over basal level in cells treated with increasing concentrations of dimeric IT (□), monomeric IT (▪), or unconjugated anti-B4 (○).
Anne-Christine Goulet et al. Blood 1997;90:
©1997 by American Society of Hematology

6. Association between increases in calcium response and sensitivity to protein synthesis inhibition by anti-B4–bR in Namalwa (•) and Daudi cells (○).
Association between increases in calcium response and sensitivity to protein synthesis inhibition by anti-B4–bR in Namalwa (•) and Daudi cells (○). (A) Increase in calcium response was expressed as the peak values (achieved at 2 to 3 minutes after IT addition) of percent increase in fluorescence over the level induced by unconjugated anti-B4 MoAb. Numbers in parentheses indicate the peak values of percentage of responding cells to anti-B4–bR over the effect of anti-B4. (B) Inhibition of protein synthesis was monitored by incorporation of 3H-leucine. Cells were incubated for 18 hours at 37°C with IT, washed twice and pulsed for 4 hours at 37°C with 1 μCi of 3H-leucine. Results are expressed as the mean cpm × 10−3 of two separate experiments, each performed in triplicate. The standard error of the mean (SEM) of each determination did not exceed 10%.
Anne-Christine Goulet et al. Blood 1997;90:
©1997 by American Society of Hematology

7. Proposed mechanisms of increased calcium mobilization induced by anti-B4–bR.
Proposed mechanisms of increased calcium mobilization induced by anti-B4–bR. (1). Homodimerization of monomeric anti-B4–bR occurs either in solution or on cell surface, leading to cross-linking of two CD19 molecules. (2a). The MoAb moiety of anti-B4–bR binds to one CD19 antigen, while a putative residual galactose binding site within the blocked ricin moiety binds to cell surface molecules. (2b). The latter interaction is specifically inhibitable with lactose. It remains to be established whether the surface molecules, which bind to blocked ricin, are CD19 or other molecules that synergize with CD19 signaling. In both models, the downstream signaling pathways may include promotion of physical interaction between CD19 and receptor-associated PTK, followed by phosphorylation of PLCγ, generation of IP3, release of calcium from intracellular stores, and influx from the extracellular milieu. The two models proposed are nonexclusive and could be complementary.
Anne-Christine Goulet et al. Blood 1997;90:
©1997 by American Society of Hematology