[Show abstract][Hide abstract] ABSTRACT: During the recall response by CD27(+) IgG class-switched human memory B cells, total IgG secreted is a function of the following: 1) the number of IgG-secreting cells (IgG-SC), and 2) the secretion rate of each cell. In this study, we report the quantitative ELISPOT method for simultaneous estimation of single-cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27(+) IgM(-) memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. BCR cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27(high) B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27(high) B cell blasts. In contrast, the addition of IL-21 to CD40L plus IL-4-activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG plus cytokine-activated B memory B cell populations, is suggestive of an on/off switch regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG-SC and the distribution of their IgG secretion rates.
The Journal of Immunology 09/2009; 183(5):3177-87. DOI:10.4049/jimmunol.0804233 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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-alpha, 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: CD19lowCD20lowCD27+CD38+HLA-DRlow. 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.
[Show abstract][Hide abstract] ABSTRACT: 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 microg/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. Fab'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.
[Show abstract][Hide abstract] ABSTRACT: Polyclonal antithymocyte globulin (ATG) is widely used as an anti-T-cell agent for induction and treatment of acute cellular rejection in solid organ transplantation. The authors recently demonstrated that rabbit (r) ATG can be used in combination with plasmapheresis to effectively treat antibody-mediated renal allograft rejection. This observation suggested that rATG may have anti-B cell activity.
The authors tested the complement-independent, apoptosis-inducing properties of rATG on CD27- naive B cells, CD40 ligand-activated B cells, and plasma cells in vitro by annexin V staining, subdiploid DNA content, caspase activation, and loss of mitochondrial membrane polarity. Potential surface targets for rATG were assayed by competitive inhibition of monoclonal antibody binding.
Rabbit ATG strongly induced apoptosis in vitro against naive, activated B cells and bone marrow resident plasma cells at clinically relevant concentrations (1-100 ng/mL). The authors found rATG activity against numerous B-cell surface proteins and observed that crosslinking of CD30, CD38, CD95, CD80, and HLA-DR likely accounts for this activity. F(ab)2 fragments of rATG showed 90% of the activity of the intact molecule, suggesting participation of the Fc fragment. Inhibition of caspase- and cathepsin-dependent apoptotic pathways partially inhibits rATG-induced B-cell apoptosis. Immunohistochemical staining of pediatric thymi demonstrated the presence of CD20+ B cells and CD138+ plasma cells within the thymic parenchyma, which accounts for the anti-B-cell activity in rATG.
Polyclonal rATG induces complement-independent apoptosis of naive, activated, and plasma B cells. This effect appears to involve the caspase- and cathepsin-mediated apoptosis pathways.
[Show abstract][Hide abstract] ABSTRACT: A major impediment to repetitive monitoring of alloreactivity or tolerance is the limited supply of donor cells available for assays of host-versus-graft T- and B-cell reactivity. In this paper, we describe the use of CD40L stimulated CD19(+) B cells as targets or stimulators in flow cytometric crossmatching (FXM), mixed lymphocyte reactivity and IFN-gamma ELISPOT assays. Stimulated B cells (sBc) express high levels of MHC class I and II, as well as the costimulatory molecules CD80 and CD86. They can be polyclonally expanded and frozen for later use. We describe the use of sBc in ELISPOT, mixed lymphocyte cultures and FXM. CD4(+) T cells exposed to sBc express a similar cytokine profile as those stimulated with unfractionated PBMC. We further analyzed T- and B-cell responses in 14 patients on the renal transplant waiting list, finding that those with an elevated panel reactive antibody (PRA) (>60%) had higher alloreactive T-cell precursor frequencies as measured by CDFSE MLR and IFN-gamma ELISPOT. We conclude that sBc are a renewable source of donor-specific target/stimulator cells for use in repetitive and coordinate assays of B- and T-cell alloreactivity.
American Journal of Transplantation 01/2005; 5(1):76-86. DOI:10.1111/j.1600-6143.2003.00637.x · 5.68 Impact Factor