Figure 1
Figure 1. Milatuzumab mediates direct cytotoxicity in CLL cells. (A) Viability of chronic lymphocytic leukemia (CLL) patient cells at 4, 24, and 48 hours by propidium iodide (PI) staining. Cells were untreated, treated with goat anti–human Fc crosslinker alone, or treated with 5 μg/mL milatuzumab (Mila) or 10 μg/mL rituximab (Ritux) in the presence or absence of Fc crosslinker (in 5 × excess of binding antibody; N = 26; P < .0001 for anti-Fc vs mila + anti-Fc). Y-axis indicates the percent of PI positive cells and the active metabolite of fludarabine (2-F-ara A; Flud) was used as a positive control for cell death. (B) Viability of CLL patient cells at 24 hours by PI staining (N = 26 from panel A; each line represents individual patient sample). (C) Immunoblots for caspases-2, -3, -8, -6, -9, and PARP in whole cell lysates isolated from CLL cells treated with anti-Fc, 5 μg/mL mila + anti-Fc, or 5μM fludarabine in the presence or absence of 20μM caspase inhibitor, Q-VD-OPH, for 24 hours. Jurkat cells treated with ultraviolet (UV) or staurosporine were used as controls for caspase cleavage. Pro-caspase (P) and cleaved (C) forms are indicated. (D) Viability of CLL patient cells by PI staining treated with anti-Fc, 5 μg/mL mila + anti-Fc, or 5μM fludarabine in the presence or absence of 20μM caspase inhibitor, Q-VD-OPH, for 24 hours (N = 5; P = .03). (Ei) Viability of CLL patient cells treated with anti-Fc alone or 5 μg/mL mila + anti-Fc in the presence or absence of stroma coculture. After 6 hours in drug, cells were washed and cultured in fresh media alone or in the presence of HS-5 stromal cells. Viability was determined by PI staining after 48 hours of coculture (N = 11; P = .10). (ii) Viability of CLL patient cells treated with vehicle or 1μM 2-F-ara A in the presence or absence of stroma coculture. After 6 hours in drug cells were washed and cultured in fresh media alone or in the presence of HS-5 cells. Viability by PI staining was determined after 48 hours of coculture (N = 6). (iii) Viability of CLL patient cells treated with anti-Fc alone or 5 μg/mL mila + anti-Fc in the presence or absence of 500 ng/mL soluble CD40L. Viability by PI staining was determined at 48 hours (N = 17; P = .11).

Milatuzumab mediates direct cytotoxicity in CLL cells. (A) Viability of chronic lymphocytic leukemia (CLL) patient cells at 4, 24, and 48 hours by propidium iodide (PI) staining. Cells were untreated, treated with goat anti–human Fc crosslinker alone, or treated with 5 μg/mL milatuzumab (Mila) or 10 μg/mL rituximab (Ritux) in the presence or absence of Fc crosslinker (in 5 × excess of binding antibody; N = 26; P < .0001 for anti-Fc vs mila + anti-Fc). Y-axis indicates the percent of PI positive cells and the active metabolite of fludarabine (2-F-ara A; Flud) was used as a positive control for cell death. (B) Viability of CLL patient cells at 24 hours by PI staining (N = 26 from panel A; each line represents individual patient sample). (C) Immunoblots for caspases-2, -3, -8, -6, -9, and PARP in whole cell lysates isolated from CLL cells treated with anti-Fc, 5 μg/mL mila + anti-Fc, or 5μM fludarabine in the presence or absence of 20μM caspase inhibitor, Q-VD-OPH, for 24 hours. Jurkat cells treated with ultraviolet (UV) or staurosporine were used as controls for caspase cleavage. Pro-caspase (P) and cleaved (C) forms are indicated. (D) Viability of CLL patient cells by PI staining treated with anti-Fc, 5 μg/mL mila + anti-Fc, or 5μM fludarabine in the presence or absence of 20μM caspase inhibitor, Q-VD-OPH, for 24 hours (N = 5; P = .03). (Ei) Viability of CLL patient cells treated with anti-Fc alone or 5 μg/mL mila + anti-Fc in the presence or absence of stroma coculture. After 6 hours in drug, cells were washed and cultured in fresh media alone or in the presence of HS-5 stromal cells. Viability was determined by PI staining after 48 hours of coculture (N = 11; P = .10). (ii) Viability of CLL patient cells treated with vehicle or 1μM 2-F-ara A in the presence or absence of stroma coculture. After 6 hours in drug cells were washed and cultured in fresh media alone or in the presence of HS-5 cells. Viability by PI staining was determined after 48 hours of coculture (N = 6). (iii) Viability of CLL patient cells treated with anti-Fc alone or 5 μg/mL mila + anti-Fc in the presence or absence of 500 ng/mL soluble CD40L. Viability by PI staining was determined at 48 hours (N = 17; P = .11).

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