B7-H1 Molecules on Myeloma Cells Induce Aggressive Cell Behavior

B7-H1 (CD274), a B7 family molecule, is expressed on antigen-presenting cells and plays crucial roles in T-cell regulation in various immune responses. We found that expression of B7-H1 molecules is also detected on some tumor cells and inhibits tumor-specific cytotoxic T lymphocytes (CTLs). Consistent with these data, it was reported that B7-H1 expression on tumor cells was associated with poor prognosis in some cancers, i.e., renal cell carcinoma, breast and ovarian cancer, etc. The expression levels of B7-H1 on plasma cells from multiple myeloma (MM) patients were reported to be higher than on those cells from patients with monoclonal gammopathy of undetermined significance (MGUS) and normal controls, suggesting that B7-H1 expression may be involved in the pathophysiology of MM. In the current study, we investigated the mechanism by which B7-H1 expression is induced on MM cells in the bone marrow (BM) microenvironment and analyzed characteristics of B7-H1⁺ MM cells in comparison with B7-H1^– MM cells, i.e., proliferative potential, drug resistance, and sensitivity to myeloma-specific CTLs and whether B7-H1⁺ MM cells are associated with patients' disease status.

We examined 14 human myeloma cell lines (HMCLs) and plasma cells from 40 MM patients, 10 MGUS patients, and 10 hematologically normal controls. B7-H1 expression levels were analyzed by flow cytometry (FCM) and real-time polymerase chain reaction (RT-PCR). Changes in B7-H1 expression levels on MM cells were analyzed after the cells were cultured in the presence of the human BM stromal cell line HS-5, its culture supernatant, various cytokines, or inhibitors of cytokines and transcription factors. Proliferative potential was compared between B7-H1⁺ and B7-H1^– MM cells, including cell cycle status analyzed by propidium iodide (PI) staining, BrdU and Ki67 expression, and cell number in liquid culture. Finally, sensitivity to dexamethasone (DEX) and melphalan (MEL) and expression of apoptosis-related genes were compared between B7-H1⁺ and B7-H1^– MM cells using FCM and RT-PCR, respectively.

1) Although B7-H1 mRNA was detected in 9 of 14 HMCLs, only 3 cell lines expressed B7-H1 on their surface in FCM. B7-H1 expression on MM cells was significantly upregulated by co-culture with HS-5 cells or their culture supernatant. 2) Among cytokines present in the HS-5 cell supernatants, i.e., interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor, and macrophage inflammatory protein 1 alpha, IL-6 alone induced B7-H1 expression on MM cells. Moreover, IL-6-neutralizing antibody dose dependently inhibited B7-H1 expression in the HS-5 cell culture supernatant. B7-H1 expression was downregulated by inhibiting STAT3, a transcription factor mediating IL-6 signaling. 3) The proliferative potential was higher in B7-H1⁺ cells in all assays. 4) Although KMS-27 myeloma cells lacking B7-H1 expression were killed by specific CTLs, KMS-27 cells that had B7-H1 expression induced on their surface resisted CTLs. 5) Nearly 20% of RPMI8226 cells expressed B7-H1, and DEX- and MEL-induced apoptosis was observed in the B7-H1^– cell fraction alone. B7-H1 gene transfection in KMS-28PE cells conferred apoptosis resistance. Compared with B7-H1^– RPMI8226 cells, B7-H1⁺ RPMI8226 cells showed higher gene expression levels of Bcl-2 and FasL and lower gene expression levels of caspase-8, caspase-9, and Fas. 6) Plasma cells from MM patients expressed significantly higher levels of B7-H1 than those cells from MGUS patients. One of 10 MM patients with International Scoring System (ISS) stage I, and 8 of 30 MM patients with ISS stage II/III were B7-H1 positive. Moreover, patients with CD45^– myeloma, who were reported to have shorter survival, had higher expression levels of B7-H1 compared with CD45⁺ myeloma patients. In 4 of 8 MM patients, B7-H1 expression levels were upregulated in relapse or refractory status compared with levels at initial diagnosis.

Our study indicated that IL-6 derived from BM stromal cells upregulates B7-H1 expression on myeloma cells, giving the cells gain more aggressive intrinsic proliferative potential and resistance to chemotherapeutic drugs and CTLs. The modulating B7-H1 pathway may have therapeutic potential in myeloma.

No relevant conflicts of interest to declare.