Abstract
Waldenstrom’s macroglobulinemia (WM) is characterized by the presence of monoclonal serum IgM and bone marrow infiltration by lymphoplasmacytoid cells. The transforming event(s) and mechanisms underlying disease progression remain uncertain. The rationale behind the use of allogeneic tumor cell lines as therapeutic vaccines is that multiple antigens shared by both the immunizing line and the patient’s tumor are presented by shared human leukocyte antigen (HLA) molecules. Most tumor cells are of poor immunogenicity and do not effectively induce antigen-specific immune responses. This is frequently due to low densities of HLA/antigen complexes and a lack of costimulatory molecules. CD80 appears to be the most efficient costimulatory molecule in humans. In this experiment, we introduced the CD80 gene into allogeneic WM cells to effect in vitro stimulation of T lymphocytes. A human WM cell line, WM103 (HLA-A2 positive, HLA-A1 and CD80 negative), kindly provided by Dr. Al-Katib, and human CD80 plasmid DNA (hB7-pBCMGHis), Dr. E. Podack, were used.
2 ×106 WM103 cells were resuspended in 100 μl of Human B Cell Nucleofector Solution (Amaxa), and 5μg of plasmid DNA. Nucleofector program U-15 used for transfection. Cells were cultured for 48h and harvested. Transfected WM103 cells were examined for expression of CD80 antigen by FACS (FACSCalibur, Becton Dickinson) using PE-conjugated anti-human CD80 (eBioscience). HLA-typed normal mononuclear cells were isolated and used as responders at a density of 1 × 106/well. Irradiated (3000 rads) WM103 cells (5 × 104/well) were used as stimulatosr at four dilutions 1:1, 1:0.5, 1:0.25 and 1: 0.125, incubated for 5 days, and treated for 18h with 1 μCi [3H] thymidine per well. liquid scintillization counting was performed in triplicate.
Successful CD80 transfection into WM103 cells. CD80 transfection into WM103 cells was confirmed by using flow cytometry.
Successful CD80 transfection into WM103 cells. CD80 transfection into WM103 cells was confirmed by using flow cytometry.
CD80 transfection induced strong T-cell proliferation in allogeeneic MLTC of heallthy donors. CD80 transfected WM103 cells can stimulate proliferation of allogeneic T-cells from adult blood.
CD80 transfection induced strong T-cell proliferation in allogeeneic MLTC of heallthy donors. CD80 transfected WM103 cells can stimulate proliferation of allogeneic T-cells from adult blood.
It has been established that T cell activation by presenting cells (APC) requires two types of signals: the first generated from T cell receptor engagement with specific antigen, and the second by constimulatory molecules. Interaction of CD80 on APCs with their natural ligand on T cells has a crucial role in costimulation and maintenance of T cell immunity. CD80 transfected allogeneic or autologous cells have not been reported in patients with WM, although similar vaccines have shown good activity in other human studies. In this report, transfection of CD80 into WM103 cells induced an allogeneic response in T cells from healthy donors, demonstrating that CD80 expression can lead to accessory cell-independent activation of native T cells. These experiments support the hypothesis that lack of expression of T-cell costimulatory molecules contributes to WM escape from immune surveillance, and provide preliminary data for the use of CD80 transfection in the immunotherapy of human WM.
Disclosure: No relevant conflicts of interest to declare.
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