Interaction Between B7-H1 Molecules on Myeloma Cells and PD-1 Molecules on T Cells Induces Resistance to Antimyeloma Chemotherapy

Introduction: B7-H1 (also known as PD-L1 or CD274), a co-inhibitory molecule of the B7 family, is detected on various tumor cells and associated with tumor evasion from cytotoxic T lymphocyte (CTL)-mediated immune surveillance. Our previous study showed that B7-H1 expression levels on plasma cells from multiple myeloma (MM) patients were significantly upregulated compared with those cells from monoclonal gammopathy of undetermined significance patients and healthy volunteers. B7-H1-expressing MM cells had a proliferative advantage and were resistant to antimyeloma agents (Tamura et al. Leukemia 2013). However, it remains unknown whether cellular responses in B7-H1-expressing MM cells are affected by the interaction of B7-H1 molecules with their receptors, i.e., PD-1 and CD80. We thus investigated the reverse signal derived from B7-H1 binding to their receptors on MM cells and examined the clinical characteristics of B7-H1-highly positive MM patients in a multicenter study.

Methods: 1) We established B7-H1-expressing MM cell lines called MOSTI expressing high levels of CD38 and CD138 from bone marrow mononuclear cells of myeloma patients and B7-H1-positive MM cells (B7-H1.KMS28PE) stably transfected with the B7-H1 gene. 2) B7-H1 knockdown MOSTI cell lines were obtained using B7-H1-specific short-hairpin RNA expressing a lentiviral vector. 3) The proliferative potential was examined by BrdU incorporation using flow cytometry (FCM) and the MTT assay. 4) Drug-induced apoptotic cells were stained with annexin V and propidium iodide (PI) and detected in FCM. 5) Magnetic Dynabeads were coupled with PD-1-Ig or CD80-Ig fusion proteins. B7-H1-expressing MM cells were co-cultured with the beads, and the binding capacity of the beads to B7-H1+ MM cells, drug sensitivity, and cell proliferation of B7-H1+ MM cells were analyzed. 6) We classified 105 cases with newly diagnosed MM into two groups according to B7-H1 expression levels on plasma cells and compared the clinical characteristics associated with prognosis between B7-H1-highly-expressing MM patients (n=43) and other patients (n=62).

Results: 1) Knockdown of B7-H1 expression in MOSTI cells significantly suppressed cell proliferation and increased melphalan-induced apoptosis. These results demonstrated that B7-H1 expression is directly associated with aggressive myeloma behavior including cell growth and drug resistance. 2) B7-H1 molecules on MOSTI and B7-H1.KMS28PE cells bound more strongly to PD-1-Fc than to CD80-Fc. The binding of PD-1-Fc to MOSTI cells was inhibited by anti-B7-H1 antibody in a concentration-dependent manner. In MOSTI cells treated with PD-1-Fc beads, apoptosis induced by both melphalan and bortezomib was markedly inhibited in comparison with the cells treated with control Ig. PD-1-Fc bead-treated B7-H1.KMS28PE cells were also resistant to melphalan-induced apoptosis. However, CD80-Fc bead-treated cells did not show drug resistance. Resistance to antimyeloma agents via the reverse signal from PD-1 to MM cells was inhibited by the PI3K/AKT inhibitor LY294002. In Western blot analysis, phospho-AKT expression was significantly upregulated in PD-1-Fc-treated MM cells. However, the cell growth of PD-1-Fc-treated MOSTI cells was the same as that of control Ig-treated cells. These data indicate that the reverse signal delivered from B7-H1 expressed on MM cells bound to PD-1 induced the drug resistance of MM cells thorough the Akt signaling pathway. 3) Patients with B7-H1 highly-expressing MM cells tended to have the poor-risk cytogenetic abnormality t(4;14) (P=0.0703). Furthermore, fibroblast growth factor receptor 3 was significantly upregulated on MM cells in those B7-H1-highly positive patients (P=0.0141). Expression levels of CD56, CD45, and CD221, which were reported to be poor prognostic markers in MM, were siginificantly higher in B7-H1-highly positive patients compared with others.

Conclusion: Our study revealed a new mechanism via which the interaction between B7-H1 on MM cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces the drug resistance of MM cells through the Akt signaling pathway. Furthermore, B7-H1 expression on MM cells may be associated with t(4;14) translocation and poor prognostic MM antigens. Thus, B7-H1 may be a reasonable target for immunotherapy.