Figure 1
CD40 stimulation induces drug resistance but sensitizes CLL cells to GA101-induced cell death. (A) PBMCs from CLL patients were cultured on 3T3 murine fibroblasts or 3T3-expressing human CD40L as described in “Methods.” After 3 days, 3T40L and 3T3 CLL cells were removed from the fibroblast layer and incubated with the indicated anti-CD20 mAbs. R indicates rituximab; G, GA101. Where indicated, cross-linking (XL) GAH Ab (50 μg/mL) was added 30 minutes after the anti-CD20 mAbs RXL and GXL. After 24 hours, apoptosis was analyzed by measuring phosphatidyl serine (PS) and annexin V exposure by flow cytometry. Top: results from individual patients (mutated: mutated IgHV genes, unmutated: unmutated IgHV genes) Bottom: averaged results from 13 CLL patients (8 mutated and 5 unmutated). Data are presented as the percentage of specific cell death, in which the background apoptosis in the absence of reagents was subtracted from the observed values in each incubation (mean ± SEM). **.001 < P < .01 and ***P < .001 significant difference from control (3T3). (B) Representative annexin V/PI FACS plots from 3T3 and 3T40L CLL cells incubated for 24 hours with XL alone, RXL and G, respectively. (C) CD40-stimulated CLL cells were incubated for 24 hours with RXL, G, and GXL, and cell death was analyzed in parallel by annexin V/PI or MitoTracker FACS staining. Stainings gave similar results in percentage of dead cells. Averaged results from 3 independent experiments (n = 7 patients; mean ± SEM). Black bars indicate dead cells as analyzed by annexin V/PI staining (% dead cells = 100% − annexin V−PI− cells). (D) Percentage of sub-G1 cells in CD40-stimulated CLL cells 24 hours after incubation with RXL, G, and GXL. Averaged results from 5 CLL patients are presented as the percentage of sub-G1 cells (mean ± SEM). *.01 < P < .05 and **.001 < P < .01 significant difference from control (medium). (E) Time course of CD40 stimulation (6, 24, and 48 hours) and subsequent incubation with RXL, G, GXL, and roscovitine. After 24 hours, apoptosis was analyzed by measuring MitoTracker signal by flow cytometry. Averaged results from 7 CLL patients are presented as percentage specific cell death (mean ± SEM). *.01 < P < .05; **.001 < P < .01. (F) CLL cells stimulated for 72 hours with 3T3, 3T40L, and CD3/CD28 activated autologous T cells (act T cell) or with soluble anti-CD40 (aCD40) were incubated with RXL, G, GXL, and roscovitine. After 24 hours, apoptosis of CLL cells was analyzed by measuring MitoTracker signal in CD2− cells by flow cytometry. Averaged results from 3 CLL patients are presented as percentage specific cell death. *.01 < P < .05; **P < .001.

CD40 stimulation induces drug resistance but sensitizes CLL cells to GA101-induced cell death. (A) PBMCs from CLL patients were cultured on 3T3 murine fibroblasts or 3T3-expressing human CD40L as described in “Methods.” After 3 days, 3T40L and 3T3 CLL cells were removed from the fibroblast layer and incubated with the indicated anti-CD20 mAbs. R indicates rituximab; G, GA101. Where indicated, cross-linking (XL) GAH Ab (50 μg/mL) was added 30 minutes after the anti-CD20 mAbs RXL and GXL. After 24 hours, apoptosis was analyzed by measuring phosphatidyl serine (PS) and annexin V exposure by flow cytometry. Top: results from individual patients (mutated: mutated IgHV genes, unmutated: unmutated IgHV genes) Bottom: averaged results from 13 CLL patients (8 mutated and 5 unmutated). Data are presented as the percentage of specific cell death, in which the background apoptosis in the absence of reagents was subtracted from the observed values in each incubation (mean ± SEM). **.001 < P < .01 and ***P < .001 significant difference from control (3T3). (B) Representative annexin V/PI FACS plots from 3T3 and 3T40L CLL cells incubated for 24 hours with XL alone, RXL and G, respectively. (C) CD40-stimulated CLL cells were incubated for 24 hours with RXL, G, and GXL, and cell death was analyzed in parallel by annexin V/PI or MitoTracker FACS staining. Stainings gave similar results in percentage of dead cells. Averaged results from 3 independent experiments (n = 7 patients; mean ± SEM). Black bars indicate dead cells as analyzed by annexin V/PI staining (% dead cells = 100% − annexin V−PI− cells). (D) Percentage of sub-G1 cells in CD40-stimulated CLL cells 24 hours after incubation with RXL, G, and GXL. Averaged results from 5 CLL patients are presented as the percentage of sub-G1 cells (mean ± SEM). *.01 < P < .05 and **.001 < P < .01 significant difference from control (medium). (E) Time course of CD40 stimulation (6, 24, and 48 hours) and subsequent incubation with RXL, G, GXL, and roscovitine. After 24 hours, apoptosis was analyzed by measuring MitoTracker signal by flow cytometry. Averaged results from 7 CLL patients are presented as percentage specific cell death (mean ± SEM). *.01 < P < .05; **.001 < P < .01. (F) CLL cells stimulated for 72 hours with 3T3, 3T40L, and CD3/CD28 activated autologous T cells (act T cell) or with soluble anti-CD40 (aCD40) were incubated with RXL, G, GXL, and roscovitine. After 24 hours, apoptosis of CLL cells was analyzed by measuring MitoTracker signal in CD2− cells by flow cytometry. Averaged results from 3 CLL patients are presented as percentage specific cell death. *.01 < P < .05; **P < .001.

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