CIK Cells Research Articles

Analysis of GCRV Pathogenesis and Therapeutic Measures Through Proteomic and Metabolomic Investigations in GCRV-Infected Tissues of Grass Carp (Ctenopharyngodon idella).

Hemorrhagic disease caused by grass carp reovirus (GCRV) infection is a major problem affecting the grass carp aquaculture industry. Therefore, inhibiting the spread of GCRV infection is of great economic significance. Herein, we sequenced five tissues (gill, liver, intestine, kidney, and muscle) from grass carp before and after GCRV infection using data-independent acquisition proteomic and untargeted metabolomic technologies, and quantitatively identified 10,808 proteins and 4040 metabolites. Then, we analyzed the differentially expressed proteins (DEPs) and metabolites (DEMs) before and after GCRV infection in the five tissues. Gene ontology analysis revealed that the five tissue DEPs were enriched in metabolic, including carbohydrate and lipid metabolic processes. Chemical taxonomy analysis showed that the categories of DEMs mainly included carbohydrates and lipids, such as fatty acids, glycerophospholipids, steroids, and their derivatives. Both the proteomic and the metabolomic data showed that GCRV affected the carbohydrate and lipid metabolism in the host. Shared pathway analysis was performed at both the protein and metabolic levels, showing significant enrichment of the glycolysis and pentose phosphate pathways (p < 0.001). Further analysis of glycolysis and pentose phosphate pathway inhibitors revealed that these two pathways are important for GCRV replication. As the kidney was the most affected among the five tissues, we analyzed the butanoate metabolism in the kidney, which revealed that most of the differentially expressed proteins and differently expressed metabolites in the butanoate metabolism were related to the TCA cycle. Further investigation showed that fumaric acid, an intermediate product in the TCA cycle, significantly inhibited GCRV replication in the CIK cells (p < 0.001), and that this inhibitory effect may be related to its induction of interferon system activation. The addition of fumaric acid to feed increased the survival rate of juvenile grass carp by 19.60% during GCRV infection, and protected the tissues of those infected with GCRV, making it a potential anti-GCRV feed additive. Our results provide new perspectives on GCRV pathogenesis and antiviral strategies for grass carp.

Immune Effect of Co-Culture of Dendritic Cells and Cytokine-Induced Killer Cells on Prostate Cancer Cells.

It was to explore the immune outcome of co-culture of dendritic cells (DC) and cytokine-induced killer cells (CIK) on prostate cancer (PCa) cells. Peripheral blood mononuclear cells (PBMCs) were extracted from healthy blood donors. DC and CIK cells were induced and co-cultured. The proliferation and phenotypic changes of DC, CIK, and DC-CIK cells/groups were observed. Model rats were constructed by injecting PC3 PCa cells into the abdominal cavity. The successful 50 cases were divided into a negative control group, a chemotherapy group, a DC group, a CIK group, and a DC-CIK treatment group (each consisting of 10 rats) to observe tumor progression. The secreted concentrations of interleukin-12 (IL-12) ((103.67 ± 2.77) pg/mL) and interferon-γ (IFN-γ) ((730.09 ± 23.52) pg/mL) were higher in DC-CIK group as against DC and CIK groups; the proliferation of CIK was higher in DC-CIK group as against CIK within 12 to 20 days of culture. The positive rate (PR) of CD3+/ CD56+ and CD8+ was higher and that of CD45RA+ was lower in DC-CIK group as against CIK.The killing rate of the DC-CIK group was higher than that of the DC and CIK groups at a target effect ratio of 10:1/20:1/50:1 (P < 0.05). After the treatment, the body weight of rats in the chemotherapy group, DC group, and CIK group was significantly reduced (P < 0.05), while no significant changes were observed in the negative control group and DC-CIK group (P > 0.05). After 25 days of treatment, the tumor size in the DC-CIK group was significantly smaller compared to the negative control group, chemotherapy group, DC group, and CIK group; the necrotic area of the tumor tissue in the DC-CIK group was also significantly larger than that in the negative control group, chemotherapy group, DC group, and CIK group (P < 0.05). Co-culture of DC and CIK is excellent in enhancing the proliferation of CIK cells, increasing the secretion of IL-12 and IFN-γ, and enhancing the activity of immune cells and anti-tumor ability, showing its potential in anti-PCa tumor immunotherapy.

New insights into zinc alleviating renal toxicity of arsenic-exposed carp (Cyprinus carpio) through YAP-TFR/ROS signaling pathway

Environmental pollution caused by arsenic or its compounds is called arsenic pollution. Arsenic pollution mainly comes from people mining and smelting arsenic compounds. In addition, arsenic compounds' widespread use and production of arsenic-containing pesticides, arsenic-rich water used to irrigate farms, or high arsenic levels in foods caused by coal burning are all sources of arsenic contamination. Arsenic contamination poses a significant threat to global public health. It is reported that exposure to arsenic can induce severe renal injury. However, the underlying mechanism needs to be clarified. In this study, the arsenic exposure model in vivo and in vitro was used to explore the mechanism of arsenic-induced renal injury, especially the role of ferroptosis and its regulatory mechanism, and then to evaluate its anti-pollution effect by supplementing zinc. The results showed that arsenic significantly induced ferroptosis, characterized by up-regulating the expression of YAP and TFR in kidney and CIK cells and then increasing the levels of Fe2+ and ROS, lipid peroxidation, and iron metabolism. Microscopic observation revealed the shrinkage of mitochondria and the increase in membrane density. In addition, molecular docking and inhibitor experiments further confirmed that arsenic is involved in the process of ferroptosis by activating YAP and TFR. These results clarify the harmful effects of arsenic on carp kidneys and its mechanism and highlight the critical interactions between the YAP-TFR pathway, ROS, and ferroptosis. Importantly, this study found that zinc can reduce ferroptosis caused by the arsenic-activated YAP-TFR pathway by inhibiting YAP activation and lipid peroxidation.

A molecular mechanism underlies grass carp (Ctenopharyngodon idella) TARBP2 regulating PKR-mediated cell apoptosis

Interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms in the innate immune system. The activated PKR performs its antiviral function by inhibiting protein translation and inducing apoptosis. In our previous study, we identified grass carp TARBP2 as an inhibitor of PKR activity, thereby suppressing cell apoptosis. This study aimed to explore the effects of grass carp TARBP2 on PKR activity and cell apoptosis. Grass carp TARBP2 comprises two N-terminal dsRBDs and a C-terminal C4 domain. Subcellular localization analysis conducted in CIK cells revealed that TARBP2-FL (full-length TARBP2), TARBP2-Δ1 (lack of the first dsRBD), and TARBP2-Δ2 (lack of the second dsRBD) are predominantly located in the cytoplasm, while TARBP2-Δ3 (lack of the two dsRBDs) is distributed both in the nucleus and cytoplasm. Colocalization and immunoprecipitation assays confirmed the interaction of TARBP2-FL, TARBP2-Δ1, and TARBP2-Δ2 with PKR, while TARBP2-Δ3 showed no binding. Furthermore, our findings suggested that the inhibitory effect of TARBP2-Δ1 or TARBP2-Δ2 on the PKR-eIF2α pathway is depressed compared to TARBP2-FL. In cell apoptosis assays, it was observed that TARBP2-FL inhibits PKR-mediated cell apoptosis. TARBP2-Δ1 or TARBP2-Δ2 exhibits decreased inhibition to PKR-mediated cell apoptosis, whereas TARBP2-Δ3 nearly completely loses this inhibitory effect. These findings highlight the critical importance of two dsRBDs of TARBP2 in interaction with PKR, as well as in the inhibition of PKR activity, resulting in the suppression of cell apoptosis triggered by prolonged PKR activation.

Preliminary study of BF/C2 on immune mechanism of grass carp against GCRV infection

BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.

Exposure to Trimethyltin Chloride Induces Pyroptosis and Immune Dysfunction in Grass Carp CIK Cells by Activating the NF-κB Pathway Through Oxidative Stress.

Trimethyltin chloride (TMT) is a highly toxic organotin pollutant frequently found in aquatic environments, posing a significant threat to the ecological system. The kidney plays a vital role in the body's detoxification processes, and TMT present in the environment tends to accumulate in the kidneys. However, it remained unclear whether exposure to different doses of TMT could induce pyroptosis and immune dysfunction in grass carp kidney cells (CIK cells). For this purpose, after assessing the half-maximal inhibitory concentration (IC50) of TMT on CIK cells, we established a model for exposure of CIK cells at varying concentrations of TMT. CIK cells were treated with various doses of TMT (2.5, 5, 10 μM) for 24 h. Oxidative stress levels were measured using kits and fluorescence methods, whereas the expression of related genes was verified through western blot and quantitative real-time PCR (qRT-PCR). The results indicated that TMT exposure led to oxidative stress, with increased levels of ROS, H2O2, MDA, and GSH, and inhibited activities of T-AOC, SOD, and CAT. It activated the NF-κB pathway, leading to the upregulation of NF-κB p65, NF-κB p50, GSDMD, NLRP3, ASC, and Caspase-1. Furthermore, TMT exposure also resulted in increased expression of cytokines (IL-18, IL-6, IL-2, IL-1β, and TNF-α) and decreased expression of antimicrobial peptides (LEAP2, HEPC, and β-defensin). In summary, exposure to TMT induces dose-dependent oxidative stress that activates the NF-κB pathway, leading to pyroptosis and immune dysfunction in grass carp CIK cells.

Emamectin benzoate exposure induced carp kidney injury by triggering mitochondrial oxidative stress to accelerate ferroptosis and autophagy

Emamectin benzoate (EMB), commonly used as an insecticide in fishery production, inevitably leaves residual chemicals in aquatic environments. High-level EMB exposure can cause severe damage to multiple systems of marine animals, potentially through mechanisms involving severe mitochondrial damage and oxidative stress. However, it is not clear yet how EMB exposure at a certain level can cause damage to fish kidney tissue. In this study, we exposed carps to an aquatic environment containing 2.4 μg/L of EMB and cultured carp kidney cells in vitro, established a cell model exposed to EMB. Our findings revealed that EMB exposure resulted in severe kidney tissue damage in carp and compromised the viability of grass carp kidney cells (CIK cells). By RNA-seq analysis, EMB exposure led to significant differences in mitochondrial homeostasis, response to ROS, ferroptosis, and autophagy signals in carp kidney tissue. Mechanistically, EMB exposure induced mitochondrial oxidative stress by promoting the generation of mitochondrial superoxide and reducing the activity of antioxidant enzymes. Additionally, EMB exposure triggered loss of mitochondrial membrane potential, an imbalance in mitochondrial fusion/division homeostasis, and dysfunction in oxidative phosphorylation, ultimately impairing ATP synthesis. Notably, EMB exposure also accelerated excessive autophagy and ferroptosis of cells by contributing to the formation of lipid peroxides and autophagosomes, and the deposition of Fe2+. However, N-acetyl-L-cysteine (NAC) treatment alleviated the damage and death of CIK cells by inhibiting oxidative stress. Overall, our study demonstrated that EMB exposure induced mitochondrial oxidative stress, impaired mitochondrial homeostasis, and function, promoted autophagy and ferroptosis of kidney cells, and ultimately led to kidney tissue damage in carp. Our research enhanced the toxicological understanding on EMB exposure and provides a model reference for comparative medicine.

Comparative Analysis of mRNA, microRNA of Transcriptome, and Proteomics on CIK Cells Responses to GCRV and Aeromonas hydrophila.

Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.

Analysis of tissue tropism of GCRV-II infection in grass carp using a VP35 monoclonal antibody

Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1–155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.

Deep sequencing identified miR-193b-3p as a positive regulator of autophagy targeting Akt3 in Ctenopharyngodon idella CIK cells during GCRV infection

Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3′-UTR of grass carp akt3, reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs.

Abstract 3595: A retrospective analysis of the clinical characteristics of metastastic solid cancer patients with cytokine-induced killer cells combined immune checkpoint inhibitor immunotherapy

Abstract Background: Cytokine-induced killer cells (CIKs), obtained through ex vivo expasion from peripheral blood mononuclear cells, are phenotypically and functionally shared with both T and NK cells. Within the T cell group, it can be classified into a group that simultaneously expresses CD3 and CD56 molecules (range: 30% to 70%) and a group representing CD3+ CD56- phenotype (range: 20% to 60%). It also contains a small population (&amp;lt; 30%) of CD3 CD56+ NK cells. They can exhibit potent MHC-unrestricted cytotoxicity for both hematologic and solid tumors, but they are not harmful in hematopoietic precursors and normal tissues. Anti-programmed death-1 (PD-1) antibodies reinvigorate T cell-mediated antitumor immunity. PD-1 blockade antibody might significantly increase the cytotoxic potency of CIK cells via increasing proliferative capacity and IFN-r secretion. This retrospective study is the first to combine CIK cells with PD-1/PD-L1/CTLA-4 blockade(Immune checkpoint inhibitors, ICIs) in patients with metastatic solid cancers. Methods: We analyzed 225 heavily treated metastastic solid cancer patients receiving CIK cell (Immunecell-LC, GC cell inc provide.) and with ICIs. Blood Sampling for CIK cells production was performed after 3 days after ICIs administration. Patients received a single intravenous infusion of activated autologous CIK cells every 2 weeks. ICIs was administered according to the protocol of each drug regardless of the CIK cells administration date. The primary end points were safety and adverse event (AE) profiles. Objective responses, overall survival (OS), progression-free survival (PFS), and diseae control rate were secondary end points. Results: Treatment-related AEs occurred in 154/225 patients. Grade 3 or 4 toxicities, including fever and chills, were observed in 28 patients. All treatment-related AEs were tolerable and no death due to autoimmune disease. When administration ICIs, CIK cells exhibited excellent antitumor properties and increased IFN-γ secretion. Objective responses (complete or partial responses) were observed in 67 of the 225 patients. One patients with hepatocellular carcionma had complete response. In most patients who showed an objective response, the effectiveness was maintained for more than 6 months. The overall disease control rate in the patients was 58.9%. At the time of this report, the median OS and PFS were 18.6 and 7.1 months in hepatocellular carcinoma patients and were 13.1 and 6.5 months in triple negative breast cancer patients, respectively. Conclusion: Treatment with activated autologous CIK cells and ICIs was safe and exerted encouraging antitumor activity in metastastic solid cancers. Phase 2 clinical studies for each tumor type will be conducted. Keywords: Cytokine-induced killer cells, Immune check inhibitors, Safety, Objective response Citation Format: Jonggwon Choi, Jong Yeup Kim, Dong Won Yang, Gun He Nam. A retrospective analysis of the clinical characteristics of metastastic solid cancer patients with cytokine-induced killer cells combined immune checkpoint inhibitor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3595.

Synergistic integration of histone deacetylase inhibitors apparently enhances the cytokine-induced killer cell efficiency in multiple myeloma via the NKG2D pathway.

The rapid recognition of epigenetic manipulation's potential in restricting cancer cell capabilities spurred translational initiatives, including histone deacetylase inhibitors (HDACis). Clinical trials on multiple myeloma (MM) demonstrated substantial benefits of HDACis, coupled with promising outcomes from cytokine-induced killer cell (CIK) immunotherapy. Intriguingly, the unexplored synergy of HDACis and CIK cell immunotherapy in MM prompted our study. We examined clinically relevant HDACis (panobinostat/LBH589 and romidepsin) alongside CIK cells derived from peripheral blood mononuclear cells across diverse MM cell lines (U266, RPMI8226, OPM-2 and NCI-H929). Utilising various in vitro methodologies, we investigated how HDACis enhance CIK cell lysis of myeloma cells through NKG2D/NKG2D ligand interactions. The results of our analysis indicated several key findings. (1) Enhanced cytotoxicity of CIK cells in MM cells when combined with HDACis. (2) Significant increase in apoptosis, suggesting HDACis and CIK may together enhance apoptotic effects in specific MM cell lines. (3) Elevated IFN-γ secretion and alterations in granzyme Bsecretion because of the independent activity of HDACis. (4) Notably, HDACis increased the expression of MICA/B and ULBP2, crucial for inducing antitumor cytotoxicity of NKTcells. Validation through NKG2D receptor blocking in CIK cells with a purified mouse antihuman NKG2D antibody further supported our findings. Our analyses provide sufficient evidence to consider this clinically forgotten instance (HDACis-CIK cell combination) as a therapeutic priority for MM treatment. Furthermore, we suggest that NKG2D/NKG2D-ligand interactions activating NK/NKTcells may contribute to enhanced myeloma cell lysis in response to HDACis treatment by CIK cells.

Polystyrene microplastics mediate cell cycle arrest, apoptosis, and autophagy in the G2/M phase through ROS in grass carp kidney cells.

Microplastics (MPs) have attracted widespread worldwide attention as a new pollutant. However, the role of reactive oxygen species (ROS) and cell cycle in nephrotoxicity induced by different concentrations of polystyrene microplastics (PS-MPs) is unknown. This study used grass carp kidney cells (CIK) treated with different concentrations of PS-MPs (0, 0.012, 0.0625, and 0.5 mg L-1 ) as subjects. With the increase of PS-MPs concentration, the levels of ROS and malonaldehyde increased, while the level of total antioxidant capacity, superoxide Dismutase (SOD), and glutathione (GSH) activity decreased. The expression of BUB1 mitotic checkpoint serine/threonine kinase (BUB1), cyclin-dependent kinase (CDK1), CDK2, CyclinB1, cell division cycle 20 homolog (CDC20), and B-cell lymphoma-2, sequestosome 1 decreased significantly. Nevertheless, the expression of Caspase 3, Cleave-Caspase 3, cytochrome c (Cytc), BCL2-associated X, apoptosis regulator, poly ADP-ribose polymerase (PARP), Cleave-PARP, Caspase 9, autophagy immunoblot kit (LC3), and Beclin1 increased. Our research shows that PS-MPs can trigger oxidative stress and induce cell cycle arrest, apoptosis, and autophagy in CIK cells by regulating ROS. This work provides a theoretical basis for cellular biology and toxicology mechanisms and new insights into the potential risks to animals from MPs exposure in the environment.

Grass carp (Ctenopharyngodon idella) deacetylase SIRT1 targets p53 to suppress apoptosis in a KAT8 dependent or independent manner

Sirtuin1 (SIRT1) is known as a deacetylase to control various physiological processes. In mammals, SIRT1 inhibits apoptotic process, but the detailed mechanism is not very clear. Here, our study revealed that grass carp (Ctenopharyngodon idella) SIRT1 (CiSIRT1, MN125614.1) inhibits apoptosis through targeting p53 in a KAT8-dependent or a KAT8-independent manner. In CIK cells, CiSIRT1 over-expression results in significant decrease of some apoptotic gene expressions, including Bax/Bcl2, caspase3 and caspase9, whereas CiKAT8 or Cip53 facilitates the induction of apoptosis. Because CiSIRT1 separately interacted with CiKAT8 and Cip53, we speculated that CiSIRT1 blocked apoptosis may be by virtue of KAT8-p53 axis or directly by p53. In a KAT8-dependent manner, CiSIRT1 interacted with CiKAT8, then reduced the acetylation of CiKAT8 and subsequently promoted its degradation. Then, CiKAT8 acetylated p53 and induced p53-mediated apoptosis. MYST domain of CiKAT8 was critical in this pathway. In a KAT8-independent manner, CiSIRT1 also inhibited p53-induced apoptosis by directly deacetylating p53 and promoting the degradation of p53. Generally, these findings uncovered two pathways in which CiSIRT1 decreases the acetylation of p53 via a KAT8-dependent or a KAT8-independent manner.

First in vitro and in vivo evaluation of recombinant IL-1β protein as a potential immunomodulator against viral infection in fish

IL-1β is an important proinflammatory cytokine with multifaceted modulatory roles in immune responses. In fish, recombinant IL-1β has been employed in the control of bacterial diseases, while the antiviral mechanisms of IL-1β remain largely unknown, and the efficacy of recombinant IL-1β as an immunomodulator to prevent viral diseases is still not determined. This study evaluated the immunomodulatory effects of recombinant grass carp IL-1β against grass carp reovirus (GCRV) in vitro and in vivo. Firstly, the mature form (Ser111-Lys270) of grass carp IL-1β was identified, and its recombinant protein (designated as rgcIL-1β) was prepared through prokaryotic expression. Then, an in vitro evaluation model for rgcIL-1β activity was established in the CIK cells, with the appropriate concentration (600 ng/mL) and effect time (1 h). In vitro, rgcIL-1β could not only induce the production of proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α but also a series of antiviral factors including IFN-1, IFN-2, IFN-γ, and ISG15. Mechanistically, transcriptome analysis and western blotting confirmed that rgcIL-1β activated multiple transcriptional factors, including NF-κB, IRF1, IRF3, and IRF8, and the signal pathways associated with inflammatory cytokines and antiviral factors expression. Expectedly, rgcIL-1β treatment significantly inhibited GCRV replication in vitro. In vivo administration of rgcIL-1β via intraperitoneal pre-injection significantly aroused an antiviral response to restrict GCRV replication and intense tissue inflammation in grass carp, demonstrating the immunomodulatory effects of rgcIL-1β. More importantly, rgcIL-1β administrated with 10 ng/g and 1 ng/g could improve the survival rate of grass carp during GCRV infection. This study represents the first time to comprehensively reveal the immunomodulatory and antiviral mechanisms of IL-1β in fish and may also pave the way for further developing recombinant IL-1β as an immunotherapy for the prevention and control of fish viral diseases.

CD19-CAR Cytokine Induced Killer Cells Armored with IL-18 Control Tumor Burden, Prolong Mouse Survival and Result in In Vivo Persistence of CAR-CIK Cells in a Model of B-Cell Acute Lymphoblastic Leukemia

Rationale: Cytokine induced killer cells (CIK) are a promising cancer immunotherapy. Recently CIK cells modified to express chimeric antigen receptors (CAR) target CD19 expressing hematological malignancies including B-cell acute lymphoblastic leukemia (B-ALL). Despite encouraging clinical trial results, insufficient CAR-CIK anti-tumor activity and persistence pose major obstacles towards improved efficacy in vivo. Therefore, we optimized our CAR-CIK platform by introducing two modifications to the CD19-CAR construct. The first modification “armored” CAR-CIK cells by inserting the IL-18 gene in a bicistronic DNA plasmid enabling simultaneous surface expression of the CAR protein and secretion of IL-18, which demonstrated improved CAR-T cell function. The second modification attenuated the CD28 cytoplasmic signaling domain of the CAR molecule to enhance anti-tumor activity and in vivo persistence since recent findings demonstrate that persistent or chronic CAR signaling can impair anti-tumor activity and in vivo persistence. Methods: CARCIK-1918 cells generated from donor-derived CIK cells are engineered to express CAR and IL-18 using the non-viral Sleeping Beauty (SB) transposon system. CIK cells were generated from donor PBMCs by the sequential addition of IFN-ϒ and stimulation with anti-CD3 antibody plus IL-2. After 2 days of stimulation CIK cells were co-electroporated with SB100X transposase mRNA and plasmid DNA containing a bicistronic transgene encoding both the anti-CD19 CAR and IL-18. This second-generation CAR contains a fusion of the CD28 transmembrane domain, intracellular CD28 and, TCR CD3ζ chain. In some instances, the cytoplasmic CD28 signaling domain is modified to attenuate CD28 signaling. This method of gene transfer results in stable surface expression of the CAR on CD3+ cells through a 17-day culture period. Day 17 cells are harvested, frozen and used for all subsequent studies. CARCIK-1918 cells are stimulated in vitro with CD19+ REH tumor cells and the phenotype of activated cells was characterized by multi-color flow cytometry. IL-18 secretion was determined by cytokine bead array analysis of supernatants collected post stimulation. CARCIK-1918 cells with or without the attenuated CD28 signaling domain were tested in vivo in a xenograft model of B-ALL, using NSG mice engrafted with CD19+ Raji tumor cells. Tumor burden was assessed weekly by bioluminescence imaging, the in vivo persistence of CARCIK-1918 cells was measured by flow cytometry, and survival was monitored throughout the study. Results: CARCIK-1918 cells express both IL-18 and CAR with or without attenuated CD28 signaling. Furthermore, stimulation of CARCIK-1918 cells with CD19+ REH tumor cells increases the frequency of CAR-CIK cells with a memory phenotype and concurrent upregulation of the high affinity IL-2 receptor, CD25, and the costimulatory receptor CD137. Expression of IL-18 leads to higher engraftment of CAR-CIK cells in tumor bearing NSG mice compared to unarmored controls. NSG mice engrafted with luciferase positive Raji cells demonstrated decreased bioluminescent signal following CARCIK-1918 treatment, consistent with CARCIK derived anti-leukemic activity. This anti-tumor activity corresponded to a statistically significant improvement in survival as mice treated with 2 nd generation CAR transgene CARCIK cells with or without attenuated CD28 signaling had a median survival of 37.5 (n=10; p=0.0001) and 38 (n=12; p =0.0001 days), respectively, compared to untreated mice that develop progressive highly disseminated tumor and succumb to disease by 3 weeks post tumor inoculation (n=11; median survival 18 days). Interestingly, mice treated with CARCIK-1918 cells with the CD28 attenuated signaling domain exhibited better tumor control and enhanced persistence in vivo, despite no significant improvement in survival compared to WT CD28 signaling CARCIK-1918 cells. Summary: This study demonstrates that administration of CAR-CIK cells armored with IL-18 results in a significant survival advantage for tumor challenged animals versus untreated animals. Furthermore, modifying the cytoplasmic CD28 domain to attenuate signaling, results in better control of tumor burden, and persistence of CARCIK-1918 cells in vivo. Our study is the first report of IL-18 armored CAR-CIK cells which may present a promising, novel strategy for the treatment of B-ALL.

MiR-22a targets p62/SQSTM1 to negatively affect autophagy and disease resistance of grass carp (Ctenopharyngodon idella)

MicroRNAs (miRNAs) are integral to many biological functions, including autophagy, a process recently proven to be closely linked to innate immunity. In this study, we present findings on miR-22a, a teleost homolog of mammalian miR-22, illustrating its capacity to target the autophagy adaptor p62, subsequently inducing downregulation at both mRNA and protein levels. Utilizing Western blot analyses, we demonstrated that miR-22a inhibits the autophagy flux of CIK cells, correlated with an elevated presence of LC3 II. Additionally, the overexpression of miR-22a resulted in the suppression of NF-κB signaling, leading to reduced cellar antimicrobial abilities and increased apoptosis. These findings provide novel insights into the role of miR-22a as an autophagy-related miRNA and its immune mechanisms against pathogens via p62 in teleost, enriching our understanding of the interplay between autophagy and innate immunity.

Selenium alleviates lead-induced CIK cells pyroptosis and inflammation through IRAK1/TAK1/IKK pathway

The toxic heavy metal lead is widely found in rivers and soils as an environmental pollutant, posing a threat to the health of aquatic organisms. Selenium is an essential trace element and a powerful antioxidant that has been shown to have anti-inflammatory and antioxidant properties as well as alleviating heavy metal poisoning. Many studies have shown that lead poisoning produces inflammatory responses and damage to the kidneys of a wide range of animals, but the effects on cellular pyroptosis and immune function and selenium antagonism in CIK cells are not clear. In this study, 500 μM Pb and 20 nM Se were applied to grass carp kidney cells, and the results showed that Pb exposure to CIK cells resulted in oxidative stress, activation of the IRAK1/TAK1/IKK pathway, up-regulation of the expression of cellular pyroptosis markers GSDMD and NLRP3, and cellular pyroptosis of CIK cells, as well as up-regulation of IL-1β and IL-18, and the generation of cellular inflammatory response. In contrast, Se treatment significantly reduced the ROS level, the expression of cellular pyroptosis markers GSDMD, NLRP3 and inflammatory element IL-1β and IL-18. Taken together, Se alleviated cellular pyroptosis and immune dysfunction caused by Pb exposure through oxidative stress and activation of the IRAK1/TAK1/IKK pathway. This study complements the harmful effects of the heavy metal Pb on fish and the real-life application of selenium in the healthy culture of fish as a reference will be provided.

Pro-inflammatory regulatory mechanism of C5a/C5aR pathway in GCRV-infected grass carp (Ctenopharyngodon idella)

Death of grass carp (Ctenopharyngodon idella) caused by grass carp reovirus (GCRV) infection severely restricts the development of the grass carp industry. The interaction of C5a and C5a receptor (C5aR) in the complement system promotes inflammation and aggravates bleeding in grass carps. To reveal the regulatory mechanism of the C5a/C5aR pro-inflammatory response after GCRV infection, the expression of C5 in the liver and muscle tissues of grass carp after GCRV infection was analyzed using immunofluorescence. The effects of over expression of C5a and the C5aR antagonist PMX205 in grass carp kidney cell line (CIK) on the phosphorylation of p38 and ERK and the gene and protein expression of IL-6 and TNF-α were also analyzed using RT-qPCR and western blot. The results showed that the expression of the C5a protein in the liver and muscle of grass carp infected with GCRV first increased and then decreased, and the C5a protein level peaked on the 5th day after infection. In CIK cells, over expression of C5a significantly increased C5aR expression, phosphorylation of p38 and ERK, and overexpression of IL-6 and TNF-α. Treatment of CIK cells with PMX205 significantly decreased the expression of C5a and C5aR, phosphorylation of p38 and ERK proteins, and expression of IL-6 and TNF-α after GCRV infection. These results implied that the interaction of C5a and C5aR in grass carp resulted in the phosphorylation of p38 and ERK proteins, a significant increase in the expression of IL-6 and TNF-α, and ultimately induction of an inflammatory response of grass carp infected with GCRV. These results provide a theoretical basis for relieving grass carp hemorrhagic diseases by regulating the interaction of C5a and C5aR.

The effect of serum origin on cytokines induced killer cell expansion and function

BackgroundCytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function.MethodsPeripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture.ResultsCell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression.ConclusionhPL at a concentration of 10% showed the best effect on CIK cell proliferation and function.