Survival of myeloma cells is critically dependent on interactions with specific bone marrow niche elements that recapitulate those that support the survival of normal bone marrow-resident long lived plasma cells (LLPC). Although the specific molecular and cellular components involved remain largely undefined, a unique characteristic of these interactions is the simultaneous direct transduction of survival signals to the myeloma/LLPC coupled with the induction from the stromal niche cells of supportive factors that further (and perhaps farther) influence the surrounding microenvironment. On the stromal side, the archetypal response is the induction of stromal IL-6 production by cell-cell contact with myeloma cells or LLPC. Although normal and malignant PC are not solely dependent on IL-6 for their survival (3
), it is clear that this cytokine is a central component of the network of soluble mediators (including APRIL/BAFF, interferons, etc (51
)) that modulate PC differentiation (which may be relevant even in myeloma in the context of the putative MM stem cell (52
)) and survival.
Thus the key pro-survival interactions are likely to involve molecules that form the signaling linkages between the myeloma/LLPC and the stromal niche. We have previously demonstrated that activation of CD28 transduces a pro-survival signal directly to myeloma cells, and now have found that myeloma cells interaction with stromal myeloid DC through CD28–CD80/CD86 engagement also induces DC production of both the supportive cytokine IL-6 and the immunosuppressive enzyme IDO. While DC production of IL-6 and IDO induced by CD80/CD86 ligation by CD28 (and CTLA4, which is not expressed on myeloma cells) has been well characterized in the context of T cell activation/inactivation, this is the first report to our knowledge that the same interaction is involved in supporting cells of the B lineage. The specific importance of this interaction is suggested by findings by us and others that CD28 overexpression on myeloma cells correlates with disease progression and most highly with the poor-prognosis MF patient subgroup. There is currently no evidence that implicates c-MAF in the direct regulation of CD28 expression (or vice versa), although c-MAF over-expression in myeloma cells enhances their integrin-mediated adhesion to BMSC (4
) and suggests a mechanism for selection of cell contact-mediated pro-survival interactions. The importance of the CD28–CD80/CD86 interaction in supporting BM-resident myeloma cells survival is further supported by our in vivo
findings in mice that B lineage knockout of CD28 (as well as global knockout of CD80 or CD86) causes a significant and selective loss of normal bone marrow LLPC without having any effect on the splenic (short lived) PC population (CHR, manuscript submitted).
Identification of CD28’s involvement in PC/MM survival points to cells bearing the CD80/CD86 ligands as components of the microenvironment, in particular macrophage/monocytes and dendritic cells which have been previously identified as being part of myeloma niche (18
). Although myeloma cells also express CD86 (but not CD80(53
)), our findings that blocking CD28–CD86 between myeloma cells alone had little effect on survival (e.g. ) suggests any pro-survival contribution of cis
interaction between myeloma cells is relatively modest. We focused on myeloid DC because they are the predominant cell type that presents CD80/CD86 to T cells, selectively infiltrate myelomatous areas in patient BM (23
) and can support PC and MM survival in vitro
). Consistent with this, we found that the myeloid compartment is increased in the myeloma bone marrow compared to normal controls, and that direct MM-DC contact protects myeloma cells against apoptosis that was significantly dependent on CD28 and CD80/CD86. Moreover, flow cytometric analysis of this CD11b+
myeloid compartment suggests that they are similar to unactivated tissue resident DC, although somewhat more mature based on their expression of CD80, CD86 and CD83. In this context it is interesting to note that the anti-myeloma agent lenalidomide, which is thought to target (currently unknown) components of the BM microenvironment, has been shown to disrupts myeloid differentiation by downregulating PU.1 in early myeloid precursors (54
However, the observation that blocking CD80/CD86 did not have the same pro-death effect in these MM-DC co-cultures as blocking CD28, at least under serum starvation conditions, suggested that CD80/CD86 were not simply acting as ligands for CD28, and that their continuing ligation by CTLA4-Ig/CD28-Ig was inducing a separate biological response. The most likely response is the induction of DC IL-6 production, which had been originally identified in the context of DC-mediated T cell activation (55
) and has not been examined in the B cell lineage. We found that MM cells can also elicit DC IL-6 production in a CD28–CD80/CD86 dependent fashion, which we believe uncovers a major mechanism by which this cytokine is elicited from the microenvironment in this disease. Unexpectedly, this induction appears to involve previously unrecognized cross-talk with the Notch1 signaling pathway. Prior studies have demonstrated that Notch receptor signaling on BMSC (which were not defined) elicits their production of IL-6, is important for the survival of MM cells that express the Notch ligand Jagged 2 (15
), and blockade of Notch signaling inhibits the ability of BMSC to protect MM cells against chemotherapy-induced apoptosis (43
). We find that when the BMSC are DC, inhibition of either the CD80/CD86 or Notch pathways abrogated DC IL-6 production to a greater extent than can be accounted for if these pathways were independent. The same crosstalk is seen when ligating CD80/CD86 with soluble CD28-Ig alone instead of with myeloma cells (CK, data not shown), indicating that these two pathways may in fact be a single integrated one.
The finding that CD28 on myeloma cells ligates CD80/CD86 to induce DC IL-6 production raised the possibility that the same ligation also induces DC production of IDO, which was also initially defined in context of DC-mediated T cell anergy and generation of Treg (30
) and has also not been examined in the B lineage. Consistent with this, CD28+
myeloma cell lines or primary myeloma isolates induced DC IDO production in a CD28 and IFNγ dependent manner. The in vivo
source of IFNγ is unclear, but it is interesting to note that pDC are a component of the pro-survival myeloma microenvironment (28
), and separately that pDC are thought to be the primary source of IFN in the context of IDO-mediated T cell tolerance (57
). Alternatively, activated T cells that infiltrate myelomatous regions of the bone marrow (58
) may also be a source of IFNγ. Our in vitro
findings demonstrate that IDO production from DC can generate both Treg () as well as directly inhibit T cell activation (although the latter only occurs at the higher levels of IDO induced when DC are cocultured with myeloma cells) (), which point to specific molecular mechanisms underlying the characteristic immunologically suppressed state seen in myeloma patients. Supportive of this, earlier clinical studies demonstrated increased kynurenine metabolites and lower tryptophan levels correlated with increased IFN activity (34
), as well as dysfunctional T cell responses with increased frequencies of regulatory T cells in myeloma patients (59
) - all pointing to an immunosuppressive role for myeloma-induced IDO that could be relieved by small molecule IDO inhibitors.
In summary, we propose that CD28 expressed by myeloma cells serves as a central molecular bridge within a complex and integrated cellular and soluble factor microenvironment necessary for MM cell survival. CD28 directly delivers a pro-survival signal to the myeloma cell, and by ligating CD80/CD86 on myeloid DC backsignals to these stromal cells to elicit both the supportive cytokine IL-6 (in conjunction with Notch1 signaling) as well as the immunosuppressive enzyme IDO. Other integral cellular components include pDC, which are induced by myeloma cells to produce APRIL/BAFF (which is complementary to IL-6 in supporting PC survival and function) and activated T cells, either of which could be the primary source of interferon required for the induction of myeloid DC IDO production. Although undoubtedly incomplete, this model begins to point to novel therapeutic targets for the treatment of multiple myeloma. In addition, we have found the interactions described here for myeloma cells very closely mirror the interactions that support the survival of normal long-lived plasma cells in the bone marrow. Thus, therapeutic inhibitors of CD28 originally developed to block T cell activation (e.g. Abatacept (CTLA4-Ig), which is FDA approved for the treatment of rheumatoid arthritis) may be effective in multiple myeloma as well as against pathogenic PC in allergic and autoimmune diseases.