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Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells.

BACKGROUND: Lack of protective antibodies and inefficient cytotoxic responses are characteristics of chronic hepatitis C infection. A defect in dendritic cell (DC) function has thus been suspected, but this remains a controversial issue. METHODS AND FINDINGS: Here we show that monocyte-derived DC (MoDC) from chronically-infected patients can mature in response to TLR1/2, TLR2/6 or TLR3 ligands. In contrast, when stimulated with the TLR4 ligand LPS, MoDC from patients show a profound defect in inducing IFNgamma secretion by allogeneic T cells. This defect is not due to defective phenotypic maturation or to the presence of HCV-RNA in DC or monocytes but is correlated to reduced IL-12 secretion by DC. Restoration of DC ability to stimulate IFNgamma secretion can be obtained by blocking MEK activation in DC, indicating that MEK/ERK pathway is involved in the Th1 defect of MoDC. Monocytes from HCV patients present increased spontaneous secretion of cytokines and chemokines, especially MIP-1beta. Addition of MIP-1beta on healthy monocytes during differentiation results in DC that have Th1 defect characteristic of MoDC from HCV patients, suggesting that MIP-1beta secretion by HCV monocytes participates in the Th1 defect of DC. CONCLUSIONS: Our data indicate that monocytes from HCV patients are activated in vivo. This interferes with their differentiation into DC, leading to deficient TLR4 signaling in these cells that are enable to induce a Th1 response. This specific defect is linked to the activation of the MEK/ERK pathway.

BACKGROUND: Hepatitis C virus (HCV) can be purified from serum of chronically-infected patients in the form of Lipo-Viro-Particles (LVP), which are triglycerid-rich lipoprotein-like particles containing viral RNA and proteins. Since LVP is a constant feature of chronically infected patients, we asked whether purified LVP could interfere with the immune response by acting directly on dendritic cell (DC) function. METHODS AND FINDINGS: We have analyzed the impact of LVP on the maturation monocyte-derived DC induced by TLR3 or TLR4 ligands. Following incubation with LVP, immature DC supported weak transient HCV-RNA replication and type I IFN synthesis. This, however, did not lead to viral particle production nor to maturation of DC. LVP-treatment prior to TLR3 stimulation by polyI:C only enhanced the secretion of IL-12, IL-6 and TNFalpha yielding typical mature DC. In contrast, LVP-treated DC activated by the TLR4 ligand LPS yielded phenotypically mature DC with reduced capacity to secrete both pro- and anti-inflammatory cytokines. Their ability to stimulate allogeneic T lymphocytes was strongly affected since activated T cells produced IL-5 and IL-13 instead of IFNgamma. Addition of IFNalpha prevented the effect of LVP on DC function. Restoration of IFNgamma secretion by T cells was obtained by blocking ERK activation in DC, while induction of IL-5 and IL-13 secretion was inhibited by blocking the p38-MAPK pathway in DC. CONCLUSIONS: LVP can interfere with TLR4-triggered maturation of DC, inducing a shift in DC function that stimulates Th2 cells instead of Th1, by a mechanism that is ERK- and p38-MAPK-dependent. The effect of LVP on DC polarization was reversed by IFNalpha, providing an additional rationale for the interferon therapy of chronically-infected patients. By acting on TLR4 pathway with LVP, HCV may thus exploit a natural protective mechanism of the liver and the intestine normally used to control inflammation and immunity to commensal microorganisms.