NX and MHL participated in the physique preparation and revised the paper. to CD14-expressing monocytes (see Additional file 1: Physique S1b for confirmation of CD14 expression). Significantly higher IL-6 levels in the monocyte culture medium were observed after 24?h of incubation with the supernatant of the effector/target coculture than immediately after beginning the incubation, which was used as the baseline level for comparison (Fig.?1c). In contrast, there were no differences in IL-6 or other cytokine levels in the monocyte culture medium after the addition of supernatants from untransduced T cells cocultured with Raji cells when comparing the same study time points. To pinpoint which cytokines are the main triggering factors that 25-Hydroxy VD2-D6 enhance IL-6 release from monocytes, we incubated primary monocytes with recombinant IL-1, IL-2, IL-6, or IFN- for 48?h, and then the released IL-6 level was measured by flow cytometry. To determine whether the enhancement in the IL-6 level is derived from activated monocytes, the amount of exogenous IL-6 was subtracted from the level measured in the monocyte culture medium. Incubation with IL-6 triggered a remarkable enhancement in the release of IL-6 from monocytes, with almost 100 times more IL-6 release in the IL-6-treated group than in the negative control group that was not incubated with any cytokines (Fig.?1d). The increases in IL-6 levels were also observed to be 25 and 3 times higher for the monocytes incubated with IL-1 or IFN- than for the negative control monocytes, respectively. In contrast, IL-2 did not trigger IL-6 release. These data demonstrated that IL-6 was the most potent cytokine in triggering monocyte IL-6 release under our experimental conditions. IL-6 knockdown does not impair basic properties of the ssCART-19 T cells To investigate whether the introduction of an IL-6-specific shRNA to regular CART-19 cells changes basic cell properties, we first designed 8 different IL-6-targeting shRNA sequences and cloned them into lentiviral vectors containing the CAR construct. IL-6 shRNA-2, which had the best IL-6 gene knockdown efficiency (70%) (Fig.?2a) and the highest IFN-/IL-6 mRNA ratio (73%) (Fig.?2b), was selected for this study. We then constructed CAR vectors containing a 4-1BB costimulatory domain, CD3 zeta domain and CD19-targeted single-chain variable fragment (FMC63) with or Klf2 without IL-6 shRNA-2 (Fig.?2c). T cells transduced with CD19-CAR were called regular CART-19 cells, and those transduced with CD19-CAR and IL-6 shRNA-2 were designated ssCART-19 cells for subsequent experiments. Open in a separate window Fig.?2 IL-6 knockdown in ssCART-19 T cells does not impair basic properties of CAR T cells. a IL-6 mRNA levels and b IFN-/IL-6 mRNA ratios in CART-19 cells expressing 8 different IL-6-specific shRNAs. c Schematic of the CD19 CAR vector containing an anti-human CD19 scFv linked to 4-1BB costimulatory domains and CD3- signaling domain with (ssCART-19) or without (regular CART-19) an IL-6-specific shRNA modification. d Transduction efficiency and the CD4/CD8 ratio of 25-Hydroxy VD2-D6 ssCART-19 and CART-19 cells. e Cell proliferation of ssCART-19 cells and regular CART-19 cells after re-stimulation with Raji cells, as analyzed by flow cytometry. f CD107a expression in ssCART-19 cells and regular CART-19 cells after induction with Raji cells. g Cytotoxicity of ssCART-19 and CART-19 cells to K562 cells, Raji cells and autologous primary acute B lymphocytic leukemia cells at different effector: target ratios. Differences among groups were assessed for significance by using one-way ANOVA. Data are shown as the mean??SD (n?=?3). NS, no significant difference, *p??0.05, **p??0.01; ***p??0.001, ****p??0.0001 Then, the basic properties of ssCART-19 cells, including transduction efficiency, the CD4/CD8 ratio after transduction, proliferation, and cytotoxicity to the corresponding cancer cells were characterized and compared with the same features of regular CART-19 cells. The ssCART-19 cells showed cell properties similar to those of the regular CAR T-19 cells in terms of the level of transduction efficiency (45.7% vs 38.1%), the ratio of CD4/CD8 (Fig.?2d), proliferation (Fig.?2e) and cytotoxicity 25-Hydroxy VD2-D6 as measured by a CD107a (Fig.?2f) degranulation assay or cytotoxicity assay when cocultured with either Raji cells or patient-derived primary B-ALL cells (Fig.?2g). Thus, the results.