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Lipopolysaccharide (LPS) leakage from bacteria causes liver inflammation and directly activates hepatic stellate cells (HSCs), contributing to liver fibrosis development. The current study investigated the effect of an autophagy inhibitor (Chloroquine, CQ) on in vitro HSC activation induced by LPS. Primary HSCs were isolated and cultured from BALB/c mice. Cells were cultured for 24 hours in a medium containing LPS 200 ng/ml, CQ 5 μM, or LPS 200 ng/ml + CQ 5 μM. All treated cells were evaluated by immunocytochemistry for morphology, proliferation, expression of activation markers α-sma and collagen 1, storage of lipid droplets and autophagy markers lc3/p62 at days 1 (D1), 3 (D3) and 7 (D7) after treatment. Proliferation of primary HSCs and expression of activation markers (α-sma and collagen 1) caused by LPS induction were significantly inhibited by CQ treatment until D7 of culture (p<0.01). Additionally, CQ treatment preserves cell function in lipid droplet storage. This effect of CQ on LPS-induced HSC activation was consistent with the late-stage inhibition of autophagy demonstrated by the decrease in lc3 and increase in p62 at D1 (p<0.01). CQ inhibits primary HSC activation in vitro when induced by LPS.

Chimeric antigen receptor (CAR) expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype composition. A higher proportion of less differentiated CAR T cells is usually associated with improved antitumoral function and persistence. We describe here a novel receptor-targeted lentiviral vector (LV), named 62L-LV, that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4 and 50% of CD8 primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered binding of 62L-LV particles to T cells nor impacted their transduction. Incubation of two days activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in an NSG mouse model. The data prove that potent CAR T cells can be generated by short-term ex vivo exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selection of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process.

Background CRS (Cytokine Release Syndrome) is considered as a recurrent side effect of CAR (Chimeric Antigen Receptor) T cell therapy. An experimental animal model for better investigating different aspects of CRS is still in demand. Besides CAR T cells`astonishing achievements, numerous efforts are determined on manufacturing hindrance, notably shortening production procedures. Here, we introduce a novel CRS mouse model employing the NSG-SGM3 strain to address adverse effects of FT-CAR T cells (Fast-Track CAR T). Methods CD19-specific FT-CAR T cells were produced from 48 hours activated human PBMC plus 24h incubation with lentiviral vectors (LV). Cytotoxic activity of FT-CAR T cells was determined against Nalm-6 cells +/-same donor-derived monocyte supplementation allowed in vitro CRS assessment. Next, to launch a CRS mouse model, FT-CAR T cells were administered into NSG-SGM3 mice engrafted with Nalm-6 cells. Mice received 1x10 7 FT-CAR or activated T cells intravenously. Health condition assessment and body index calculation were regularly monitored using weight, and body temperature measurements. Human cytokines in plasma were determined by the LEGENDplex kit.Results Flow cytometric analysis revealed that the majority of FT-CAR T cells were positive for the VSV-glycoprotein. Upon reactivation and further cultivation, 65% of all T cells converted into CAR-positive T cells by day 6. The FT-CAR T cells indicated significant cytotoxic activity against tumor cells compared to T cell control, without any contraction in presence of monocytes. The co-culture supernatant displayed significantly elevated amounts of pro-inflammatory cytokines, including IL-6, INF-g, TNF-a, and IL-10. Next, we appointed the NSG-SGM3 mice to refine FT-CAR T cell competence in CRS induction. While all control mice were in good condition, detrimental side effects came up rapidly within 24h for all FT-CAR T-treated mice. Tremendous temperature change over 2°C and more than 10% weight loss led to termination of this group. Highly elevated cytokine levels were observed, notably enhanced for IFN-g, TNF-a, IL-2, IL-10 (all P<0.0001), and IL-6 (P<0.0121). Conclusions Our study introduces an appropriate CRS mouse model to substantiate the acute side effects of FT-CAR T cells.The key feature of this model is innate myeloid cells releasing cytokines upon interaction with CAR T cells. Our results suggest that FT-CAR T cells carry residual LV components and can induce at least as severe CRS as conventional CAR T cells.

Chimeric antigen receptor (CAR) T cells have emerged as effective strategy against B cell malignancies. Since the long manufacturing process limits patient accessability, short-term (st) CAR T cells are under investigation. Here, we evaluated CD19-CAR T cells 24 hours after exposure to lentiviral vectors. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and strong release of CRS-relevant cytokines (IL-6, IFN-γ, TNF-α, GM-CSF, IL-2, IL-10). When administered into tumor engrafted NSG-SGM3 mice, severe acute adverse events encompassing high body scoring, temperature and weight drop arised rapidly within 24 hours. Human (IFN-γ, TNF-α, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines typical for severe cytokine release syndrome (CRS) were systemically elevated. Our data highlight potential safety risks of CAR T cells manufactured within short time and suggest simple models for their preclinical safety evaluation.