Characterization of B cell responses in relation to organ transplantation
Heidt, S.
Citation
Heidt, S. (2010, March 3). Characterization of B cell responses in relation to organ transplantation. Retrieved from https://hdl.handle.net/1887/15051
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Chapter 5
Bortezomib affects the function of human
B cells: possible implications for humoral
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Bortezomib inhibits B cell activation
5
INTRODUCTION
The proteasome inhibitor bortezomib is a potent inducer of apoptosis in malignant (1) as well as non-malignant human plasma cells (2). For that reason, bortezomib has recently come to the attention of the transplantation community for the treatment of humoral rejection. Bortezomib was shown to reduce donor specific antibody levels and effectively revert rejection episodes (3, 4), as well as attenuate HLA antibody levels in non-rejecting transplant patients (5). Since proteasomes are present in all eukaryotic cells, bortezomib, besides its effects on plasma cells, likely affects other cell types of the immune system as well. Additional drug effects towards B cells may be beneficial for the treatment of humoral rejection. Therefore, we tested whether, in addition to plasma cell depletion, bortezomib was capable of inhibiting the functionality of human B cells upon CD40 or B cell receptor mediated stimulation.
MATERIALS AND METHODS
Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll Hypaque density gra- dient centrifugation from blood of healthy blood bank donors after informed consent. B cells were immunomagnetically isolated by positive selection (Invitrogen, Leek, the Neth- erlands). These CD19+ B cells were cultured in Iscove’s Modified Dulbecco Medium (Gib- co, Paisley, UK) supplemented with 10% FCS (Gibco), 0.05 mM 2-mercaptoethanol (Sigma- Aldrich, Zwijndrecht, the Netherlands) and ITS (Sigma-Aldrich) at 37°C and 5% CO2. B cells were activated with 1 µg/mL of anti-CD40 mAb (R&D systems, Minneapolis, MN, USA), 100 U/mL of IL-2 (EuroCetus, Amsterdam, the Netherlands), 25 ng/mL IL-10 (R&D systems), 100 ng/ml of IL-21 (Invitrogen) and 2.5 µg/ml of the TLR9 ligand CpG ODN 2006 (Hycult Biotechnology, Uden, the Netherlands), or with 1:10.000 diluted Staphylococcus
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RESULTS AND DISCUSSION
Bortezomib completely abrogated IgM and IgG production as well as proliferation in a dose dependent fashion. Complete inhibition of immunoglobulin production was achieved at 1.0 ng/ml of bortezomib with IgM and IgG levels equally influenced (Figure 1a). Prolif- eration was already completely inhibited at 0.75 ng/ml of bortezomib. Additionally, when added to B cells that were already activated for 2 days, bortezomib completely inhibited proliferation, albeit at slightly higher doses (Figure 1b). Bortezomib also inhibited immu- noglobulin production and proliferation of Staphylococcus aureus stimulated B cells (data not shown), indicating that this is a general effect of bortezomib on B cells, regardless of the mode of stimulation.
We conclude that, in addition to depleting plasma cells, bortezomib profoundly abrogates B cell function. Thus, bortezomib may prevent the de novo emergence of HLA antibodies as well as deplete an important source of antigen presenting cells. Our findings suggest that, when bortezomib is used for desensitization or for the treatment of humoral rejec- tion, there is no need for additional anti-B cell therapy, such as anti-CD20 mAb treatment.
Figure 1. Bortezomib effectively inhibits immunoglobulin production and proliferation of activated human B cells. Iso- lated B cells were cultured at 5×103 cells per well in 96-wells round bottom plates for 7 days in the presence of graded concentrations of bortezomib. At day 6, supernatants were collected for immunoglobulin (Ig) detection. Bortezomib dose-dependently inhibited IgM and IgG levels in B cell cultures (a). After 7 days of culture, B cell proliferation was mea- sured by 3H-TdR incorporation. Bortezomib added either directly (t= 0 h), or after 2 days (t= 48 h) dose-dependently inhibited B cell proliferation (b). Data are depicted as means of at least triplicate wells with standard deviation. Repre- sentative data from one experiment are shown; similar results were obtained in four independent experiments. CPM:
counts per minute.
Bortezomib inhibits B cell activation
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REFERENCES
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