Versikine acts through IRF8 to promote transcription of ISGs. (A) IRF transcription in MM1.S cells, following 48 hours of coculture with THP-1 macrophages in the presence of 0.5 μM versikine (Vkine) or vehicle (veh). Expression is normalized to veh-only levels at 4 hours. (B) IRF9 mRNA levels in MM1.S cells cocultured with THP-1 macrophages in the presence of 0.5 μM versikine (gray bars) or vehicle (black bars) for indicated time lengths. (C) THP-1 cells expressing Vkine or an empty-vector (EV) control were transduced with control lentivirus (NT) or lentivirus expressing short hairpin (shRNA) targeting IRF8 (shIRF8) (see supplemental Figure 3 for validation of IRF8 knockdown at the protein level). Versikine-mediated induction of 3 ISGs shown was measured in the presence and absence of IRF8. (D) EBI3 transcription in MM1.S cells cocultured with THP-1 macrophages and treated with 0.5 μM versikine (gray bars) or vehicle (black bars) for indicated time lengths. (E) RT-PCR analysis for EBI3 transcripts in patient-derived, freshly explanted MAM treated with 1 μM versikine for 12 hours. Relative expression is normalized to vehicle-only control (= 1). (F) Staining of human myeloma bone marrow core biopsy consecutive sections with antibodies against neoepitope DPEAAE generated by V1-VCAN cleavage at Glu⁴⁴¹-Ala⁴⁴² and T-cell marker CD8. DPEAAE constitutes the C terminus of versikine. Four patterns of staining were observed in 19 informative punches: Pattern (a): intense/moderate VCAN proteolysis-CD8 infiltration/aggregates (>5 CD8⁺ cells in cluster). Pattern (b): intense/moderate VCAN proteolysis-CD8 poor (single/doublet CD8⁺ cells sparsely distributed within tumor). Pattern (c): weak/focal VCAN proteolysis-CD8 poor (single/doublet CD8⁺ cells sparsely distributed within tumor). Pattern (d): absent VCAN proteolysis-CD8 poor (single/doublet CD8⁺ cells sparsely distributed within tumor). (G) Proposed immunomodulatory roles of VCAN proteolysis in the myeloma microenvironment. Whereas intact VCAN is thought to exert tolerogenic activities through TLR2 binding on antigen-presenting cells, its proteolytic product, versikine, may promote immunosurveillance through IRF8-mediated effects on antigen-presenting cells and tumor cells. Currently untested hypotheses are represented by broken lines. *P < .05, **P < .01, ***P < .001, ****P < .0001.