Multiple myeloma (MM) is a neoplasm of bone marrow (BM) resident long-lived plasma cells (LLPC) that comprises 20% of all hematologic malignancies - second only to non-Hodgkin’s lymphoma. Despite new chemotherapeutic agents, MM remains incurable. Although initially sensitive to chemotherapy, the primary cause of treatment failure is the development of progressively more chemo-refractory disease whose resistance is due to upregulation of pro-survival mechanisms. Thus defining the specific mechanisms of myeloma cell survival/drug resistance is essential for the development of curative treatment approaches in MM. It is now clear that myeloma cells are critically dependent on interactions with BM microenvironment (ME) for their survival, just like their normal LLPC counterparts, and these interactions play a major role in MM resistance to chemotherapy. Despite their central importance in myeloma survival however, the specific molecular and cellular components involved in these interactions remain poorly characterized. In our original proposal, we hypothesized that CD28 was playing significant pro-survival role in myeloma. Although it has been primarily characterized as the prototype T cell costimulatory receptor involved in T cell activation and survival, CD28 is also expressed on normal PC and MM. Work accomplished during the last funding cycle has demonstrated that CD28 activation through two downstream signaling pathways plays an essential role in the survival of both normal LLPC and MM cells, and that therapeutically blocking CD28 activation leads to MM cell death/resensitization to chemotherapy in preclinical in vivo models. Based on these findings and initial evidence for clinical efficacy, we have just opened a phase II clinical trial of blocking CD28 activation in refractory/relapsed MM (NCT02334865) More recently, we have found that CD28 activation substantially enhances the metabolic fitness of MM cells, which plays a major role in its pro-survival effect. In addition to directly supporting MM survival, we have found that MM CD28 also modulates the MM ME. CD28 engagement of its ligands CD80 and CD86 on stromal dendritic cells (DC) initiates CD80/CD86 “backsignaling” that induces DC production of the pro-MM survival cytokine IL-6 and the immunosuppressive tryptophan (Trp)-catabolizing enzyme indoleamine 2, 3 dioxygenase (IDO). We have also shown that CD86 expressed on MM cells has independent pro-survival function. More recently we have determined that kynurenine (Kyn, produced by IDO degradation of Trp) is an endogenous ligand for the aryl-hydrocarbon receptor (AhR), and AhR activation has a significant but previously unrecognized pro-survival role in MM. The Specific Aims of this proposal are: 1). Characterize the effect of blocking CD28 activation on myeloma chemoresistance in a Phase 2 clinical trial in MM patients, 2). Define how CD28 activation enhances the metabolic fitness of myeloma cells, and 3). Determine the role of MM CD28-media...