Specific cytometric bead array

To assess short-term cytotoxic activity we used a standard ⁵¹Cr release assay [44 (link)]. Chromum-51 labeled CD138⁺ and CD138⁻ target cells were incubated with T cells at different effector to target ratios, and in medium alone or in 1% Triton X-100 (Sigma-Aldrich) to determine spontaneous and maximum ⁵¹Cr-release, respectively. The mean percentage of specific lysis of triplicate wells was calculated as follows: [(test counts − spontaneous counts)/(maximum counts − spontaneous counts)] × 100%. Coculture experiments were also performed. Control and CD138.CAR-Ts were cocultured in 24-well plates in the presence of CD138⁺ or CD138⁻ target cells (at 1:1 E:T ratio) and in the absence of exogenous cytokines. Phenotypic analyses were performed on day 3 or 5 of coculture and T cells and tumor cells detected using CD3, CD138, or CD19 Abs. We also cocultured T cells with autologous primary MM cells and with endothelial or epithelial cells. In the latter case, CD31 Ab was used to identify endothelial cells. Co-culture supernatant was harvested after 24 hours of culture and cytokines measured using specific cytometric bead arrays (BD), or specific ELISAs (R&D System) according to manufacturer's instructions. The soluble CD138 was also assessed in the culture supernatant of tumor cells using a specific ELISA kit (R&D System).

Release of IFN-αs was measured by a specific ELISA kit (PBL assay science). Production of cytokines (IL-6, TNF-α, IL-1β, IL-10 and IL-12p70) and chemokines (CXCL-10, CCL-2, CXCL-9, CXCL-8 and CCL-5) was quantified by specific cytometric bead arrays (BD Biosciences) on a FACS Canto (BD Biosciences) and analyzed by FCAP array software (BD Biosciences).