B7.2 and B7-H2 Molecules Stimulate Proliferation of Myeloma Cells and Inhibit Anti-Myeloma Immune Responses.

The B7 family molecules, which play an important role in immune responses by costimulating or coinhibiting T cells via antigen-T-cell receptor interactions, are expressed not only on professional antigen-presenting cells but also on some tumor cells. We reported that B7.2 and B7-H2 molecules in acute myeloid leukemia had immunomodulatory functions and were associated with poor prognosis (Clin Cancer Res, 2005). In this study, we investigated whether these B7 family molecules are expressed on myeloma cells, and if myeloma cells expressing the molecules have a functional advantage for survival. First, we investigated the expression of B7.2 and B7-H2 molecules on B cell lineage cells in various maturation stages in normal bone marrow (BM). Although B7.2 was consistently expressed on pre-B, mature B, and plasma cells, the B7-H2 expression was relatively high on pre-B and mature B cells and extremely low on plasma cells. When we examined the expression on human myeloma cells using flow cytometry (FCM), 7 and 9 of 14 myeloma cell lines expressed B7.2 and B7-H2 molecules, respectively. The percentages of B7.2-positive cells in CD38^high plasma cells in BM were much higher in myeloma patients (n=35) than those in with monoclonal gammopathy of unknown significance (MGUS) (n=12) (mean±SD: 40.0±30.2 vs 20.6±19.5). Extremely high expression of B7-H2 was detected in 3 myeloma patients but in no MGUS patient. The expression of B7.2 and B7-H2 on myeloma cells was induced or enhanced by cultivation with autologous stroma cells or cytokines such as tumor necrosis factor (TNF)-α, a growth-promoting cytokine in myeloma. Furthermore, the percentages of cells in G0/G1 phase were lower and those in G2/M phase higher in the B7.2 (or B7-H2)-positive population compared with the B7.2 (or B7-H2)-negative population in a KMS-27 myeloma cell line. We isolated B7 molecule-positive and B7 molecule-negative KMS-27 cells using a FACS Vantage FCM (BD Biosciences) for further investigation. B7.2 (or B7-H2)-positive cells proliferated more than B7.2 (or B7-H2)-negative cells in vitro. Cells expressing B7.2 and B7-H2 induced allogeneic CD4⁺ T cells to proliferate and secrete interleukin (IL)-10 but not interferon-g in vitro, and these effects were partially blocked by antagonistic antibodies to B7.2 and B7-H2. In separate experiments, IL-10 increased the proliferation of myeloma cell lines and inhibited anti-myeloma cytotoxic T cells. We performed transwell studies, in which KMS-27 cells were placed in the upper chamber and CD4⁺ T cells with irradiated KMS-27 cells in the lower chamber with or without blocking antibodies to B7.2 or B7-H2. The results confirmed that B7.2 or B7-H2 molecules on KMS-27 cells induce CD4⁺ T cells to produce a soluble factor(s) that stimulates KMS-27 cell proliferation. Taking the results together, B7.2 and B7-H2 molecules on myeloma cells:

1. have expression induced by TNF-α;

2. are associated with actively cycling cells; and

3. induce normal CD4⁺ T cells to produce a soluble factor such as IL-10 that stimulates proliferation of myeloma cells and inhibits anti-tumor immune responses.

Therefore, these molecules may be involved in the pathophysiology in myeloma.