Apoptosis and complement-mediated lysis of myeloma cells by polyclonal rabbit antithymocyte globulin

Current monoclonal antibody therapies for multiple myeloma have had limited success, perhaps due to narrow target specificity. We have previously described the ability of polyclonal rabbit antithymocyte globulin (rATG) to induce caspase- and cathepsin-mediated apoptosis in human B and plasma cells. We now extend this observation to myeloma cells. Complement independent cell death was measured after addition of rATG (1-1000 μg/mL) to cultures of myeloma cell lines or primary CD138+ isolates from patient bone marrow aspirates. rATG induced significant levels of apoptosis in myeloma cells as assayed by caspase induction, annexin V binding, subdiploid DNA fragmentation, plasma-membrane permeability, and loss of mitochondrial-membrane potential. Addition of complement greatly augmented myeloma-cell death. Binding of rATG to individual myeloma cell-surface proteins, primarily CD38, CD52, CD126, and CD138, was demonstrated by competitive inhibition experiments with targeted monoclonal antibodies. Three pathways of cell death were identified involving caspase activation, cathepsin D, and the genistein sensitive tyrosine kinase pathway. F(ab′)2 fragments of rATG had reduced proapoptotic activity, which was restored by coincubation with Fc fragments, and anti-CD32 or anti-CD64 antibodies. We conclude that rATG is an effective agent for in vitro induction of apoptosis in multiple myeloma, and that exploratory clinical trials may be warranted

CpG DNA activation and plasma-cell differentiation of CD27(−) naive human B cells

Unmethylated CpG DNA activation of naive CD27(−) B cells has been reported to require B-cell–receptor (BCR) cross-linking. We describe a culture system using CpG DNA with sequential steps for T-cell–independent activation of naive CD19(+)CD27(−) human peripheral blood B cells that induces efficient CD138(+) plasma-cell differentiation. CD27(+) and CD27(−) B cells were cultured in a 3-step system: (1) days 0 to 4: CpG, IL-2/10/15; (2) days 4 to 7: IL-2/6/10/15 and anti-CD40L; (3) days 7 to 10: IL-6/15, IFN-α, hepatocyte growth factor, and hyaluronic acid. Both CD27(+) and CD27(−) B cells up-regulated intracytoplasmic TLR-9 following CpG DNA activation. CD27(−) B-cell activation required cell-cell contact. Both naive and memory B cells progressed to a plasma-cell phenotype: CD19(low)CD20(low)CD27(+)CD38(+)HLA-DR(low). Seventy percent of the CD27(−)-derived CD138(+) cells demonstrated productive V chain rearrangements without somatic mutations, confirming their origin from naive precursors. Plasma cells derived from CD27(+) B cells were primarily IgG(+), while those from CD27(−) B cells were IgM(+). Our results indicate that under certain conditions, naive B cells increase TLR-9 expression and proliferate to CpG DNA stimulation without BCR signaling. In addition to its immunologic significance, this system should be a valuable method to interrogate the antigenic specificity of naive B cells