B-lymphocyte stimulator (BLyS) stimulates immunoglobulin production and malignant B-cell growth in Waldenstrom macroglobulinemia

Abstract

Waldenström macroglobulinemia (WM) is a serious and frequently fatal B-cell malignancy associated with an elevated monoclonal IgM protein in the serum. Many of the mechanisms leading to this disease are not yet known. B-lymphocyte stimulator (BLyS) is a TNF family member that is critical for maintenance of normal B-cell development and homeostasis. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B cells. It also regulates immunoglobulin secretion by normal B cells. To determine the relevance of BLyS in WM, we examined the role of BLyS in WM patient samples. Malignant B cells were found to bind soluble BLyS and variably express the receptors BAFF-R, TACI, and BCMA. We also found expression of BLyS in bone marrow specimens by immunohistochemistry and elevated serum BLyS levels in patients with WM. BLyS, alone or in combination with cytokines that induce immunoglobulin production, was found to increase IgM secretion by malignant B cells. Furthermore, BLyS was found to increase the viability and proliferation of malignant B cells from WM patients. Due to the role of BLyS in WM, strategies to inhibit BLyS may potentially have therapeutic efficacy in these patients.

Figures

Figure 1.

Expression of BAFF-R, BCMA, and TACI on WM cells. Tumor cells from the WM cell line (WSU-WM) and WM patients were stained with biotin-conjugated BLyS, anti-BAFF-R, anti-BCMA, or anti-TACI for 30 minutes at 4°C, then washed and incubated for 30 minutes with PE-streptavidin. Isotype and fluorochrome controls were done in parallel with each sample (white histograms). Gray histograms correspond to CD19+CD138+ cells from a representative example of each subtype. Results shown are from 5 representative WM samples, out of a total of 12 patient samples analyzed.

Figure 2.

BLyS expression in bone marrow specimens of WM patients. Immunohistochemical analysis of BLyS expression in 3 WM bone marrow specimens (WM 6-WM 8) was performed using anti-BLyS mAb as described in “Materials and methods.” A similar analysis was performed on a normal bone marrow specimen (NBM). Original magnification for images shown: × 600.

Figure 3.

Elevated BLyS levels in the sera of healthy and WM patients. Serum BLyS levels were analyzed by ELISA in specimens obtained from healthy individuals (n = 38) and from WM patients (n = 9). Results are presented as individual values of each patient, and the bar represents the value of the mean.

Figure 4.

Elevated IgM levels in the presence of BLyS. CD19+CD138+ cells (0.25 × 106 cells/well) from WM patients (n = 5) were cultured in 48-well plates in the presence of 100 ng/mL BLyS, cytokines (20 U/mL IL-2, 5 ng/mL IL-6, 5 ng/mL IL-10, and 2 ng/mL IL-12), a combination of BLyS + cytokines, or media alone. One week following treatment, supernatants were harvested and used to test for IgM secretion using ELISA. Results for 5 WM patients are presented as means ± standard deviation (SD) of duplicate wells.

Figure 5.

BLyS promotes survival of WM cells. CD19+CD138+ cells (0.25 × 106 cells/well) from WM patients (n = 4) were cultured in 48-well plates in RPMI 10% FBS in the presence or absence of 100 ng/mL BLyS. One week following treatment, the cells were harvested and assayed for viability using annexin-V/PI staining. Cell viability in the presence of BLyS was normalized to the media-alone control and is presented as relative viability.

Figure 6.

BLyS promotes proliferation of WM cells. (A) CD19+CD138+ cells (0.1 × 106 cells/well) from WM patients (n = 3) were cultured in 96-well round-bottom plates in the presence or absence of 100 ng/mL BLyS or 10 μg/mL anti-Ig Ab, each alone or in combination, for 5 days at 37°C in the presence of 5% CO2. Values represent the mean of triplicate values ± SD. (B) TACI-Ig fusion protein blocks BLyS-induced proliferation of malignant B cells. A fourth WM patient sample (WM15) was used to show that BLyS-induced proliferation of malignant B cells is inhibited in the presence of TACI-Ig fusion protein.