Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients

Abstract

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Figure 1

Ultrastructural changes in plasma cells incubated with bortezomib. A. Normal plasma cells in control samples. Note the typical morphology, with elaborate endoplasmic reticulum (ER) and eccentric nuclei (with a cart-wheel heterochromatin configuration) B. Most plasma cells also appeared largely normal after 2 hours of treatment with 2.5 μM bortezomib. C. After 8 hours of treatment with bortezomib, most plasma cells appeared apoptotic, with heterochromatin condensed around the perimeter of the nucleus and distension of the ER lumen. After 24 hours with bortezomib, no plasma cells could be detected. Coded cell samples were post-fixed with osmium tetroxide and counter-stained with uranyl acetate and lead citrate. Pictures are representative of the conditions analyzed. Scale bars are 2 μm.

Figure 2

Changes in plasma cell numbers and functions after addition of bortezomib (2.5 μM), lenalidomide (10 μM) or dexamethasone (10 nM) on days 7 and 11, and assessed 3 days later. A. Representative plasma cells from cytocentrifuged thymic cells. They were readily identified by their staining for internal IgG (green) and surface CD138 (red; DNA is blue), and typical morphological features, including: relatively large size, abundant cytoplasm with positive staining for immunoglobulins and eccentric nuclei. However, many plasma cells showed dim or negative staining for CD138 despite their intense labeling for internal IgG and typical morphological features (37). They were frequently found in clumps of 2-3 or more cells together, as shown in the lower panel. Scale bars are 10 μm. B. Normalized plasma cell numbers on day 14. Each point represents a single well (3 per condition) from the indicated patients; horizontal bars are median values. Results are expressed as percentage of the corresponding control cultures of the same patient, from which the absolute plasma cell numbers are given in Table 2. C. Spontaneous secretion of AChR Abs in vitro, measured by radio-immunoprecipitation and expressed as nanomoles per liter per day (AChR Ab production rate). Each point represents the average of samples from patients MG-1 - MG-3 (4 replicates per patient). Samples from day 14 could not be compared to earlier time points because of inter assay-variation, and are therefore not shown. D. AChR Ab production rate on day 11. Each point represents a measurement of one well (4 per condition) from the indicated patients; horizontal bars are median values. No AChR Ab production was detected in samples of MG-6. E. Spontaneous secretion of total IgG in vitro. We measured IgG in supernatants by ELISA, and expressed results as nanograms of IgG per milliliter per day (IgG production). F. Total IgG production rate on day 14. Each point represents one well (4 per condition) from the indicated patients; horizontal bars are median values. Results are normalized as for Fig 2B, and absolute IgG production rates shown in Table 2. Arrows indicate the days of drug addition. Error bars correspond to the SEM. One-way (B, D, F) or two-way (C, E) ANOVA and Bonferroni post-hoc testing were used for statistical analyses. Lenalidomide was not tested for MG-8.

Figure 3

Susceptibility of irradiated cells to experimental drugs. Thymic cells were irradiated and subsequently cultured for two weeks. Drugs were added on days 7 and 11 (bortezomib - 2.5 μM, lenalidomide −10 μM, dexamethasone - 10 nM) and samples were collected and analyzed as for Fig. 2. A. Plasma cell survival from patients MG-1, 5 and 6 on day 14 shown as % of the absolute numbers in the control cultures (Table 2). B. AChR autoAb secretion from patient MG-1 on day 11. The AChR autoAb production rate from patients MG-3, 5 and 6 was below detection limit; in MG-2, it was just above background. C. IgG production rate from irradiated samples on day 14. Each point represents one well (3 per condition) from the indicated patients; horizontal bars are median values. One-way ANOVA and Bonferroni post-hoc testing were used for statistical analyses.

Figure 4

Quantitative overview of the drug effects on B- and T-lymphocytes. Thymic cells were cultured for 2 weeks and drugs were added on days 7 and 11 (indicated by arrows). Cells were collected and labeled for FACS analysis on days 0, 2, 9 and 14. Samples were gated to exclude cell debris, selected for viable (PI-negative) cells and for either CD3 or CD19. In, A, C and E, each point represents the average of samples from patients MG-1, MG-2 and MG-3 (3 per patient). Error bars correspond to the SEM. Each point in panels B, D and F represents one well (3 per condition) from the indicated patients for day 14; horizontal bars are median values. Results are normalized as for Fig 2B, and absolute cell numbers are shown in Table 2. At least 10,000 total events per sample were recorded; numbers of events analyzed per gate and per patient are given in Supplemental Table S1. One-way ANOVA and Bonferroni post hoc testing were used for statistical analyses.

Figure 5

Dose-response curves for plasma cell numbers (A), AChR Ab production (B) and total IgG secretion (C), after addition of drugs on day 4 and assay on day 7. Thymic cells from patients MG-1 and MG-3 were used in A; cells from MG-4 and MG-5 in B; and cells from MG-1, MG-3, MG-4, MG-5, MG-7 and MG-8 in C. Each point represents the average of the normalized results for all the patients analyzed (at least 3 replicates per patient) and error bars the SEM. One-way ANOVA and Bonferroni post-hoc testing were used for statistical analyses. *** p<0.001, compared with the untreated cultures (white square).