Cytokine-Induced Killer Cells

Three-dimensional tumor spheroids were generated with a single spheroid per well [57 (link)] and automatically imaged using a Celigo cell cytometer (colony counting embryoid body application). To generate the tumor spheroids, we used ultra-low attachment (ULA) 96-well round-bottom plates (Corning Incorporated, Corning, NY, USA) without additional coating. After adding 200 μL of target cell suspension (cell density, 25 x 10³ target cells/mL) to each well, we centrifuged the culture plates with an absolute number of 5000 target cells per well at 1000 g for 10 minutes. Half of the culture medium was replaced every 3 to 4 days. On day 4 of tumor spheroid culture, we added 200,000 WT or ErbB2-CAR CIK cells to each well. The culture conditions were visualized 6 hours later and every 24 hours thereafter. On day 10 of co-culture, the tumor spheroids and effector cells were harvested and used for further analysis.

pCMV-CMV-GFP was employed as original plasmid (28 (link)) for constructing the following expression plasmids: LGP2-Flag, RIG-I-Flag, MDA5-Flag, RIG-I-HA, RIG-I-CARD-HA, RIG-I-Helicase-HA, RIG-I-RD-HA, MDA5-HA, MDA5-CARD-HA, MDA5-Helicase-HA, MDA5-RD-HA, RIG-I-CARD-Flag, and MDA5-CARD-Flag. The ORFs or the domains of the relevant genes were amplified from grass carp spleen tissue cDNA and then inserted behind the first CMV promoter. The Flag or HA tag was introduced by the reverse primer. The primers were listed in Table S1 in Supplementary Material. To construct the luciferase reporter vectors of grass carp IPS-1, MITA, IRF3, IRF7, IFN1, IFN2, IFN3, IFN4, IFNγ1, IFNγ2, NF-κB1, and NF-κB2, the 5′-flanking fragments of these genes were obtained from the grass carp genome (37 (link)). The core promoter regions were predicted by WWW Promoter Scan,¹ GPMiner,² and Promoter 2.0 Prediction Server.³ To verify the promoter activities, the predicted promoters of the related genes were introduced to the pCMV-GFP vector by replacing the CMV promoter (38 (link)). The primers were shown in Table S1 in Supplementary Material. Then, these vectors were transfected into CIK cells by FuGENE 6 Transfection Reagent (Promega), respectively. The promoter activity was reflected by promoting green fluorescent protein expression, observed under a fluorescence microscope (Nikon). The promoter activities of RIG-I, MDA5, and MITA were verified in the previous studies (39 (link)–41 (link)). For dual-luciferase reporter assay, the valid promoters were cloned into pGL3-basic luciferase reporter vector (Promega), respectively. For transient transfection, CIK or FHM cells were plated in 24-well plates, 6-well plates, or 10 cm² dishes with 70–90% confluency. Approximately 24 h later, transfection was performed with FuGENE 6 Transfection Reagent (Promega) according to the manufacturer’s instructions. LGP2 stable transfected cell line was obtained by G418 selection as previously reported (38 (link)). It is worth noting that the vector (pCMV-CMV-EGFP) used for protein overexpression in the present study contains two CMV promoters, which promote the expressions of target protein and EGFP, respectively, and the later is used to monitor the transfection efficiency. Hence, we employed empty vector-transfected cells rather than normal cells as control in the present study, which can make a better demonstration of LGP2 functions in RLR signaling pathways, not EGFP or other components in the vector skeleton. To assess the influence of empty vector on dual luciferase reporter assay, transcription level, and protein expression, we compared the promoter activities, mRNA expressions, and protein levels between empty vector-transfected cells and normal cells, and the results demonstrated that empty vector has no significant influence on the promoter activity, mRNA level, and protein synthesis (Figures S1 and S2 in Supplementary Material; Figure 3E).

Centers were requested to report patients receiving cellular therapies other than HCT. Hematopoietic advanced cellular therapies were defined as infusion of cells undergoing substantial manipulation after collection, either selection and/or expansion, or genetic modification and thus qualify as investigational or approved advanced therapy medicinal products (ATMPs) according to Regulation (EC) N° 1394/2007. In this context, “substantial” should be understood as referring to the definition included in the Regulation and subsequent regulatory documents and may not reflect the workload assumed by cell processing facilities working in conjunction with clinical programs. Depending on their nature and indications, hematopoietic cellular therapies may be designed to replace or to complement HCT. Administration of non-substantially manipulated hematopoietic cells, such as transplantation of CD34+ selected hematopoietic stem cells were counted as HCT and not as cellular therapy [18 (link)]. Similarly, un-manipulated lymphocyte infusions post-HCT were counted as donor lymphocyte infusions (DLI) and not as cellular therapy. Hematopoietic cellular therapies include immune effector cells as defined in FACT-JACIE standards for Hematopoietic Cellular Therapy: “A cell that has differentiated into a form capable of modulating or effecting a specific immune response.” This definition covers CAR-T cells and forms the basis for accreditation requirements in recent EBMT-JACIE recommendations [17 , 19 (link)].
Hematopoietic cellular therapies were categorized as chimeric antigen receptor T cells (CAR -T); in vitro selected/and or expanded T cells or cytokine activated, such as virus specific T cells; cytokine-induced killer cells (CIK); regulatory T cells (TREGS); genetically modified T cells other than CAR-T; natural killer cells (NK); dendritic cells; mesenchymal stromal cells; in vitro expanded CD34+ cells; and genetically modified CD34+ cells. This survey did not include cells from sources other than hematopoietic tissue. On the other hand, gene therapy protocols, such as those used to treat thalassemia or SCID were included, however numbers have remained low.

The renal carcinoma cell lines (786-0 and ACHN) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured according to a standard protocol. FDX1 overexpression and negative control plasmids were synthesized by Shanghai GeneChem Co., Ltd. We utilized Lipofectamine iMAX to transfect cell lines with plasmids and harvested infected cells via puromycin culture for 3 weeks. CCK8 kits were adopted to test the proliferation difference between the control and FDX1 over expression treated ccRCC cell lines. We collected peripheral venous blood from healthy volunteers and stored it in a heparin anticoagulant tube. We then added Ficoll lymphocyte separation solution into the tube to dilute the blood. The PBMC were retrieved from the isolated lymphocytes. The recombinant human IFN-γ, IL-2, and CD3 monoclonal antibodies were added into PBMC suspended in serum-free medium to obtain cytokine-induced killer cells (CIK). Activated CIK were cocultured with FDX1-overexpression or control ccRCC lines for 32 h at a ratio of 6:1. The collected supernatant was collected for the enzyme-linked immunosorbent assay (ELISA) to quantify IFN-γ and IL-2 production (R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. Absorbance was detected at 490 nm via a BioTek microplate reader. For the detailed procedure of the experiments, refer to our previous studies [29 (link),30 (link),31 (link),32 (link)].

We collected PBMCs samples form adult HCC patients for the expansion of CIK cells. All of the HCCs were diagnosed by cytological or pathological evaluations, or according to the criteria of the American Association for the Study of Liver Disease [27 (link)]. We excluded patients with severe alcoholic hepatitis or acute or chronic liver failure, non-HBV, HCV, or alcohol-related HCC, and patients with concurrent evidence of sepsis.