Silencing or blockade of CD86 results in myeloma cell death. (A) Myeloma cell lines were infected with lentiviral particles carrying empty vector (pLKO.1) or the individual shRNAs, and cell death was monitored by annexin V–fluorescein isothiocyanate staining for 4 days. Data for different time points were all compared with pLKO.1 controls (left). mRNA quantification as measured via qRT-PCR comparing levels of CD28 or CD86 to vector-controls (middle). Representative histograms show CD28 or CD86 surface levels at day 4 postinfection. Thin gray histograms at left are isotype controls (right). (B) mRNA quantification as measured via qRT-PCR comparing levels of CD28 or CD86 with vector controls (left). Representative histograms show CD28 or CD86 surface expression at day 4 postinfection. Thin gray histograms at left are isotype controls (right). (A-B) All data are presented as mean ± standard error of the mean (SEM) of at least 3 independent experiments. *P < .05, **P < .01, ***P < .005. All qRT-PCR data are normalized to β-actin as an endogenous control and then compared relative to mRNA levels in pLKO.1 empty vector control. RNA was extracted on day 3 postinfection. For flow cytometry data, histograms are representative of the annexin V⁻ set in the population. (C) Gating strategy for cells from the buffy coat from a bone marrow aspirate of a patient with myeloma. Total cells were separated into CD138⁺ (purple) vs CD138⁻ (orange). Histograms show that CD138⁺ cells are also CD38⁻ CD28⁻ CD86⁺. (D) Cells from the buffy coat from the same patient with myeloma were infected with lentivirus containing shCD86 or pLKO.1 empty vector control. Cell death of CD38⁺ vs CD38⁻ cells was assessed via staining with annexin V at indicated time points postinfection. Representative histograms for CD86 surface expression are from day 3 postinfection. FSC, forward scatter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SSC, side scatter.