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Interferon-Gamma Induces CD20 Expression on Multiple Myeloma Cells via Induction of Pu.1 and Augments Rituximab Binding to Myeloma Cells

Interferon-Gamma Induces CD20 Expression on Multiple Myeloma Cells via Induction of Pu.1 and Augments Rituximab Binding to Myeloma Cells

Rituximab (Rituxan) therapy is successfully used to treat many B-cell malignancies. Absent or diminished CD20 expression on certain B-cell tumors may limit the efficacy of CD20-directed serotherapies.

We attempted to identify agents that could induce CD20 expression on these B-cell tumors. We first screened RPMI 8226 multiple myeloma cells with agents known to modulate lymphoid-cell surface receptors, and identified interferon-gamma (IFN-gamma [Actimmnue]) as an inducer of CD20 expression.

We subsequently cultured multiple myeloma patient plasma cells (N = 20), multiple myeloma patient B cells (N = 9), chronic lymphocytic leukemia (CLL) patient B cells (N = 5), non-Hodgkin’s lymphoma (NHL) B cells (N = 3), normal donor B cells

(N = 11), normal donor plasma cells (N = 3), and normal donor CD34-positive hematopoietic progenitor cells (N = 5) without and with IFN-gamma (1 to 100 U/mL) for 48 hours, and evaluated changes in CD20 expression with multicolor flow cytometry. Rituximab binding to RPMI 8226 multiple myeloma cells and multiple myeloma patient plasma cells was also determined before and after IFN-gamma treatment.

These studies demonstrated that IFN-gamma induced CD20 expression (increased mean intensity, and percentage of cells expressing CD20) on multiple myeloma plasma cells, multiple myeloma B cells, and normal donor plasma cells. In contrast, CD20 expression on CLL, NHL (CD20low), and normal donor B cells, as well as on normal donor progenitor cells, was unaffected by IFN-gamma.

The levels of IFN-gamma receptor expression alone did not explain the above differences, since a comparable percentage of multiple myeloma (82% ± 6%) and CLL B cells (88% ± 9%) expressed the IFN-gamma receptor. However, IFN-gamma receptor expression was lower on normal donor (26% ± 6%) vs multiple myeloma and CLL B cells (P < .001). Rituximab binding to RPMI 8226 cells and to multiple myeloma patient plasma cells increased following culture with pharmacologically attainable levels of IFN-gamma (1 to 100 U/mL).

Changes in Pu.1 expression, a transactivator of CD20 expression that is downregulated with plasma cell differentiation, were also examined to discern the means by which IFN-gamma induces CD20 on plasma cells. Western blot analysis and im-munofluorescence staining of RPMI 8226 plasma cells treated with IFN-gamma for 0 to 48 hours showed that Pu.1 was induced at 6 hours, and coincided with CD20 upregulation.

CONCLUSION: IFN-gamma induces CD20 on multiple myeloma plasma cells and multiple myeloma B cells and augments rituximab binding to multiple myeloma plasma cells. These findings provide the rationale for use of rituximab with CD20-directed serotherapies for multiple myeloma.

Click here for Dr. Bruce Cheson’s commentary on this abstract.

 
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