Preferential Induction of B Cell Apoptosis Using Photodynamic Therapy.

Apoptotic cell death in cancer cells promotes antigen presentation and favors the development of an anti-tumor vaccination effect. While lymphoma cells can easily be obtained from lymph nodes or peripheral blood, these malignant cells usually co-localize with normal hematopoietic T lymphocytes, rendering the selective induction of apoptosis in malignant B cells particularly difficult. We have found that photodynamic therapy (PDT) using the photosensitizer 4–5 dibromorhodamine methyl ester (TH9402) and visible light (514 nm) can be used to preferentially eliminate malignant B-lineage non-Hodgkin’s lymphoma cells (mean of 3–4 logs) over T lymphocytes. As a rhodamine derivative, TH9402 cellular efflux is mediated through the P-glycoprotein (Pgp) pump. However, the preferential elimination of B cells over resting T cells could not be ascribed to Pgp inactivation and we found no difference in intracellular retention of TH9402 between B and T cells. In order to understand the molecular events leading to such high B cell susceptibility to PDT, we then investigated the nature of death pathways involved. To determine whether apoptosis was the preponderant mechanism leading to the rapid elimination of B cells, we measured the binding of Annexin V (AnnV) in 7-aminoactinomycin D (7AAD) negative (i.e. live) B and T cells using flow cytometry, and quantified DNA fragmentation and caspase 3 and 9 activity in isolated CD3+ and CD19+ PDT treated and untreated cells. We found that B cells demonstrated high levels of apoptosis after PDT, with 53±1.3% (mean±SE) AnnexinV+/7AAD- cells compared to 30±1.9% in T cells (p< 0.05). The extent of DNA fragmentation, as determined by the TUNEL reaction, was extensive in B cells (33.7±9.8%)(mean±SE) but not T cells (2.3±0.1%) when measured at 4 hours post PDT (p<0.01). After PDT, catalytic caspase 3 levels were also 4-fold higher in CD19+ cells than CD3+ cells (p<0.01). In contrast, caspase 9 levels remained low for both cell types. The induction of apoptosis translated into the elimination of more than 90% of B cells within 4 hours after PDT, while the majority of T cells were not affected. Importantly, similar elimination of B-lineage cell lines and patient cells was observed, using a limiting dilution assay, whether cells overexpressed the anti-apoptotic bcl-2 protein or not. Thus, these results indicate that the predominant caspase 3 apoptotic pathway is specifically implicated in PDT-induced B cell death, and its early activation explains the rapid and profound sensitivity of B lymphocytes to PDT. This strategy could promote tumor cell vaccination in the context of autologous TH9402-purged stem cell transplantation for patients with B-lineage malignancies. In addition, preferential B cell targeting could take advantage of the crucial immunomodulatory role of apoptosis for the treatment of patients with autoimmune B cell disorders.