IFN-γ Promoter Research Articles

A “Prime and Expand” strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming (“Prime” step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 (“Expand” step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting “Prime and Expand” ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the “Prime and Expand” strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression

Despite the remarkable success of CAR T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous IFN-γ promoter for transgenic IL-15 expression. We demonstrate that IFN-γ expression is tightly regulated by TCR signaling. By introducing IRES-IL15 into the 3’-UTR of the IFN-γ gene via HDR-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen-stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Lastly, as part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

The Correlation Between IFNG Gene Methylation and Th1|Th2 Cell Balance in ROU and the Interventional Study of Jiaweidaochi Powder.

The pathogenesis of recurrent oral ulcers (ROU) is complex, with a long duration of illness and challenging to cure. According to traditional Chinese medicine (TCM),"heat accumulation in the heart-spleen" is one of the main causative factors. Jiaweidaochi powder (JWDCP) is based on the ancient Chinese medicine formula JWDCS, with the addition of Tongcao and gypsum and the removal of Mu Tong. It is generally used to treat "heat accumulation in the heart-spleen." Previous studies have demonstrated that it effectively reduces recurrence rates and is anti-inflammatory in modulating immunity. The ROU rats' model for JWDCP intervention treatment had been established, and histological tests revealed that JWDCP has a therapeutic effect on the pathological changes in the oral mucosa. In addition, the methylation levels of peripheral blood IFNG gene were detected by bisulfite sequencing PCR (BSP), and the methylation levels of the IFNG promoter region in the model group and each dose group were lower than those in the control group. However, no significant methylation differences were observed. Furthermore, the results of enzyme-linked immunosorbent assay (ELISA) and RNA quantitative polymerase chain reaction showed that JWDCP could reduce IFN-γ and IL-4 protein concentrations, with high GATA-3 mRNA production, T-bet mRNAproduction was upgraded,elevated IL-4 mRNA levels, and reduced IFN-γ mRNAlevels after treatment (P < 0.001). The expression of transcription factor T-betmRNA and GATA-3 gene mRNA was accompanied by changes in IFN-γmRNA and IL-4mRNA, demonstrating that Th2 type differentiation in RAS suppresses the body's immunity and that the imbalance of transcription factor expression further leads to Th1/Th2 drift. JWDCP is likely to reduce the protein concentration by regulating the imbalance of transcription factors and enhancing antioxidant capacity, thus achieving therapeutic effects. Treatment of recurrent oral ulcer models is not sufficient to reset IFNG methylation levels, correlating with the refractoriness of ROU, further confirming the complexity of epigenetic mechanisms and that epigenetic alterations in specific mediators may persist locally.

Association of IFN-γ +874 A/T SNP and hypermethylation of the -53 CpG site with tuberculosis susceptibility.

Tuberculosis (TB) is now the 2nd leading infectious killer after COVID-19 and the 13th leading cause of death worldwide. Moreover, TB is a lethal combination for HIV-patients. Th1 responses and particularly IFN-γ are crucial for immune protection against Mycobacterium tuberculosis infection. Many gene variants for IFNG that confer susceptibility to TB have been described in multiple ethnic populations. Likewise, some epigenetic modifications have been evaluated, being CpG methylation the major epigenetic mark that makes chromatin inaccessible to transcription factors, thus avoiding the initiation of IFNG transcription. We evaluated both genetic and epigenetic changes involved in IFN-γ production and TB susceptibility in Argentine population. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed for the IFN-γ +874 A/T polymorphism (rs2430561) genotyping in 199 healthy donors (HD) and 173 tuberculosis (TB) patients. IFN-γ levels from M. tuberculosis-stimulated PBMCs were measured by ELISA. The methylation status at the -53 CpG site of the IFNG promoter in individuals with latent infection (LTBI), TB and HD was determine by pyrosequencing. Using a case-control study, we found that A allele and, consequently, AA genotype were overrepresented in patients with active disease. Moreover, HD carrying T allele (AT or TT genotype) evidenced an augmented IFN-γ secretion compared to TB patients. Codominance was the genetic model that best fits our results according to the Akaike information criterion (AIC). In addition, increased methylation levels at the -53 CpG site in the IFN-γ promoter were observed in whole blood of patients with active TB compared to LTBI individuals. IFN-γ is regulated by genetic variants and epigenetic modifications during TB. Besides, AA genotype of the rs2430561 single nucleotide polymorphism could be considered as a potential TB susceptibility genetic biomarker in Argentina and the methylation of the -53 CpG site could result in a useful predictor of TB reactivation.

MiR-16-5p specifically inhibits IFN-γ-regulated memory T helper cell differentiation in malignant pleural effusion of non-small cell lung cancer.

The mechanism for memory T helper (Th) cell differentiation in malignant pleural effusion (MPE) of non-small cell lung cancer (NSCLC) is poorly understood. MicroRNAs (miRNAs), as small non-coding RNA that regulate gene expression, play a crucial role in the regulation of memory Th cell differentiation. However, whether miRNAs can inhibit the differentiation of memory Th cells in MPE of NSCLC has not been reported. This study aimed to explore miR-16-5p specifically inhibits interferon-gamma (IFN-γ)-regulated memory Th cell differentiation in MPE of NSCLC. A total of 30 patients with NSCLC and 30 age- and sex-matched patients, who were clinically diagnosed as benign pleural effusion (BPE) of lung disease and had not received any intervention, were collected. The expression of nucleic acids, miRNAs, and cytokines was detected by polymerase chain reaction (PCR), miRNA microarray, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and western blotting. The expression of CD4+CD69+ T cells in NSCLC with MPE was lower than that in lung disease BPE. CD4+CD69+ T cells highly express CD45RO+ and mainly secrete anti-tumor cytokines IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α). The expression of miR-16-5p in CD4+CD69+ CD45RO+ T cells in MPE was higher than that in BPE. Moreover, miR-16-5p can bind to both IFN-γ promoter and its 5'untranslated region (5'-UTR), suggesting that IFN-γ may be the target gene directly affected by miR-16-5p. IFN-γ also affects the differentiation of memory CD4+ T cells by regulating T-bet. We believe that miR-16-5p may regulate the decrease of differentiation of naïve CD4+ T cells into memory CD4+CD69+ T cells through its target gene IFN-γ in MPE, thus reducing the number of cytokines that produce anti-tumor effects. It may be the main reason for the low response rate of lung cancer with MPE immunotherapy.

Dust fall PM2.5-induced lung inflammation in rats is associated with hypermethylation of the IFN-γ gene promoter via the PI3K-Akt-DNMT3b pathway

Inflammation is one of the major adverse effects of fine particulate matter (PM2.5) on the lung system; however, its mechanisms remain unclear. Rats were exposed to different concentrations of PM2.5 to investigate the mechanism of short-term exposure-induced lung inflammation. The regulation of PI3K-Akt and DNA methyltransferase 3b (DNMT3b) was assessed by using a PI3K inhibitor and a DNA methyltransferase inhibitor. We found that PM2.5 could decrease interferon-γ (IFN-γ) levels and increase interleukin 4 (IL-4), IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF) to promote eosinophil infiltration and eventually lead to allergic pulmonary inflammation. Moreover, the CpG island methylation rate of the IFN-γ promoter and the protein expression of DNMT3b, PI3K and p-Akt were increased in lung tissues after PM2.5 exposure. Both inhibitors reversed the CpG island hypermethylation of IFN-γ. In conclusion, in PM2.5-induced lung injury, the activated PI3K-Akt pathway, via an increase in DNMT3b expression, is involved in CpG hypermethylation of the IFN-γ gene promoter.

A feeder cell-free activation and expansion strategy to generate memory-like NK cells sufficient for off-the-shelf multi-dose adoptive cell therapy.

Abstract Adoptive cell therapy (ACT) using NK cells is a promising armament in the fight against cancer. Cytokine induced memory like (CIML) NK cells have been shown in clinical studies to have potent antitumor activity with superior in vivo persistence. Currently, the expansion of NK cells for clinical development is mainly based on feeder cells, which imposes significant regulatory hurdles and increases the costs for manufacturing. We have developed fusion proteins, HCW9201 and HCW9206 comprising of IL-15/IL-18/IL-12 and IL15/IL-7/IL-21, respectively, capable of priming memory-like differentiation and expanding CIML NK cell products without using feeder cells. This “Kick and Expand” strategy allows greater than 100x expansion of CIML NK cells from donor PBMCs in as little as 14 days without the use of exogenous feeder cells. Continued expansion can yield sufficient CIML NK cells for cryopreservation and multiple ACT infusions. The NK cells generated have bona fide memory-like properties: enhanced antitumor activity across multiple cancer cell lines, higher metabolic capacity, stable epigenetic demethylation of the IFN-γ promoter and increased persistence in NSG mice, when compared to conventional NK cells. In conclusion, this “Kick and Expand” process supports generation of abundant CIML NK cells for multiple ACT infusions and provides simpler, more regulatory friendly, off-the-shelf platform for generating NK cell products, including those with chimeric antigen receptor (CAR) constructs.

Identification and functional characterization of interleukin-12 receptor beta 1 and 2 in grass carp (Ctenopharyngodon idella)

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12Rβ1) and IL-12Rβ2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12rβ1 (gcIl-12rβ1), gcIl-12rβ2a and gcIl-12rβ2b. In silico analysis showed that gcIl-12rβ1 and gcIl-12rβ2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12rβ2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12rβ1 and gcil12rβ2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12rβ2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12rβ1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12rβ1 being genuine orthologues of mammalian IL-12Rβ1. Moreover, our data showed that gcIl-12rβ1 and gcIl-12rβ2a but not gcIl-12rβ1 and gcIl-12rβ2b mediated the effects of rgcIl-12 isoforms on ifn-γ promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.

Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores.

The T-box transcription factor Eomesodermin (Eomes) promotes the expression of interferon-γ (IFN-γ). We recently reported that the small molecule inhibitors, TPCA-1 and IKK-16, which target nuclear factor κB (NF-κB) activation, moderately reduced Eomes-dependent IFN-γ expression in mouse lymphoma BW5147 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In the present study, we investigated the direct effects of NF-κB on IFN-γ expression in mouse lymphoma EL4 cells and primary effector T cells. Eomes strongly promoted IFN-γ expression and the binding of RelA and NFATc2 to the IFN-γ promoter when EL4 cells were stimulated with PMA and IM. Neither TPCA-1 nor IKK-16 reduced IFN-γ expression; however, they markedly decreased interleukin (IL)-2 expression in Eomes-transfected EL4 cells. Moreover, TPCA-1 markedly inhibited the binding of RelA, but not that of Eomes or NFATc2 to the IFN-γ promoter. In effector CD4+ and CD8+ T cells activated with anti-CD3 and anti-CD28 antibodies, IFN-γ expression induced by PMA and A23187 was not markedly decreased by TPCA-1 or IKK-16 under conditions where IL-2 expression was markedly reduced. Therefore, the present results revealed that NF-κB is dispensable for IFN-γ expression induced by PMA and calcium ionophores in EL4 cells expressing Eomes and primary effector T cells.

Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 Tcell effector differentiation.

SUMMARYTo gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression. IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human chimeric antigen receptor T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector DNA demethylation programming in CD8 T cells and serve as proof of concept that cytokines can guide induction of epigenetically regulated traits for T cell-based immunotherapies.

Physical exercise increases global and gene-specific (interleukin-17 and interferon-γ) DNA methylation in lymphocytes from aged women.

What is the central question of this study? Is lymphocyte DNA methylation differentially modulated by resistance training and aerobic exercise in older women? What is the main finding and its importance? The practice of resistance training led to an increased global DNA methylation in lymphocytes. The exercise-induced increase of inflammatory genes methylation may be associated with immune function impairment during ageing. Ageing-induced increase in inflammatory gene expression through a reduction in DNA methylation might contribute to chronic diseases. Regular physical exercise practices, in turn, are associated with a decrease in the incidence of inflammatory diseases. We herein evaluated the effects of three exercise modalities on lymphocyte global and gene-specific (interferon γ (IFN-γ) and interleukin 17A (IL-17A) DNA methylation in aged women (68±7.5years). This cross-sectional study included 86 women, divided into four groups according to the physical exercise practice: 20 were practicing resistance training (RT); 24 were practicing water aerobics exercise (W); 22 were practicing water aerobics and resistance exercise (RWT), and 20 did not practice any physical exercise (CON). We evaluated volunteer functional capability using the Timed Up and Go (TUG) test, global lymphocyte DNA methylation by enzyme-linked immunosorbent assay, IFN-γ and IL-17A methylation by qPCR and CD4+ IFN-γ+ and CD4+ IL-17+ cell percentage by flow cytometry. The three physically exercised groups performed functional capability tests in a shorter period and showed a higher global lymphocyte DNA methylation and methylated CpGs of IL-17A and IFN-γ promoter regions than the control group. The practice of resistance training (RT and RWT groups) lead to high global DNA methylation. The combination of resistance training and aerobic exercise led to the increase of lymphocyte IL-17A and IFN-γ gene methylation induced by each separately. However, the percentage of IFN-γ+ and IL-17+ cells was lower only in the RT group. The exercise-induced increase of inflammatory-gene methylation may be associated with gene expression changes and immune function impairment during ageing.

Function and mechanism of histone demethytransferase Jmjd3 mediated regulation of Th1/Th2 balance through epigenetic modification in pre-eclampsia

Objective: To observe the expression level of histone demethyltransferase Jmjd3 in patients with pre-eclampsia (PE), and to investigate the possible mechanism of its epigenetic modification in regulating Th1/Th2 imbalance in PE patients. Methods: The mRNA levels of histone demethyltransferase Jmjd3 from peripheral blood mononuclear cells (PBMC) of PE patients and normal pregnant women were detected by RT-PCR. Peripheral serum IFN-γ and IL-4 were detected by ELISA. RT-PCR was used to detect the mRNA levels of Jmjd3, Tbx21 and Cxcr3 in the spleen of PE and control mice. Immunomagnetic beads were used to sort out the initial CD4+ T cells in the spleen of control and PE mice. Western blot was used to detect H3K27me1 and H3K27me3 levels. ChIP analysis was used for H3K27me3 demethylation modification in spleens of PE mice. Results: Compared with normal pregnant women, the mRNA level of Jmjd3 in PBMC of PE patients was significantly increased, the level of IFN-γ in serum was significantly increased, and the level of IL-4 was significantly reduced (P<0.01). Compared with normal control mice, the mRNA level of Jmjd3 in the spleen of PE mice was significantly increased, and the expression of Tbx21 and Cxcr3 was significantly increased in PE mice (P<0.01); the H3K27me3 level of CD4+ T cells in PE mice was significantly reduced (P<0.05), but H3K27me1 was not changed. ChIP analysis showed that CD4+ T cells H3K27me3 in PE group mice were in the Ifng promoter region, compared with control mice. Recruitment was significantly reduced, while recruitment in the promoter region of Il4 was significantly increased (P<0.01). Conclusions: In both PE patients and mice with PE model, the relative expression level of histone demethyltransferase Jmjd3 is significantly up-regulated, which further induces the demethylation of H3K27me3 in the Ifng promoter region and promotes the initial CD4+ T cells to Th1 cell differentiation and development, leading to an imbalance of Th1/Th2, which may be one of the important reasons for the development of preeclampsia.

Host Genetic Variants Potentially Associated With SARS-CoV-2: A Multi-Population Analysis.

BackgroundClinical outcomes of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) showed enormous inter-individual and inter-population differences, possibly due to host genetics differences. Earlier studies identified single nucleotide polymorphisms (SNPs) associated with SARS-CoV-1 in Eastern Asian (EAS) populations. In this report, we aimed at exploring the frequency of a set of genetic polymorphisms that could affect SARS-CoV-2 susceptibility or severity, including those that were previously associated with SARS-CoV-1.MethodsWe extracted the list of SNPs that could potentially modulate SARS-CoV-2 from the genome wide association studies (GWAS) on SARS-CoV-1 and other viruses. We also collected the expression data of these SNPs from the expression quantitative trait loci (eQTLs) databases. Sequences from Qatar Genome Programme (QGP, n = 6,054) and 1000Genome project were used to calculate and compare allelic frequencies (AF).ResultsA total of 74 SNPs, located in 10 genes: ICAM3, IFN-γ, CCL2, CCL5, AHSG, MBL, Furin, TMPRSS2, IL4, and CD209 promoter, were identified. Analysis of Qatari genomes revealed significantly lower AF of risk variants linked to SARS-CoV-1 severity (CCL2, MBL, CCL5, AHSG, and IL4) compared to that of 1000Genome and/or the EAS population (up to 25-fold change). Conversely, SNPs in TMPRSS2, IFN-γ, ICAM3, and Furin were more common among Qataris (average 2-fold change). Inter-population analysis showed that the distribution of risk alleles among Europeans differs substantially from Africans and EASs. Remarkably, Africans seem to carry extremely lower frequencies of SARS-CoV-1 susceptibility alleles, reaching to 32-fold decrease compared to other populations.ConclusionMultiple genetic variants, which could potentially modulate SARS-CoV-2 infection, are significantly variable between populations, with the lowest frequency observed among Africans. Our results highlight the importance of exploring population genetics to understand and predict COVID-19 outcomes. Indeed, further studies are needed to validate these findings as well as to identify new genetic determinants linked to SARS-CoV-2.

Safety and Efficacy of an Immune Cell-Specific Chimeric Promoter in Regulating Anti-PD-1 Antibody Expression in CAR T Cells

T cells modified to co-express cytokine or other factors with chimeric antigen receptor (CAR) can induce substantial and persistent increases in antitumor capacity in vivo. However, the uncontrolled expression of cytokines or factors can lead to the overactivation of immune cells, causing severe adverse events such as cytokine release syndrome (CRS) and neurotoxicity by CAR T cells with excessive growth potential. Conventional promoters are unregulated, and their expression is unlimited in T cells. In this study, by connecting the cytomegalovirus (CMV) enhancer, core interferon gamma (IFN-γ) promoter, and a T-lymphotropic virus long terminal repeat sequence (TLTR), we constructed and screened the chimeric promoter CIFT, which was highly expressed in some cell lines secreting IFN-γ and silenced in others. We placed this promoter upstream of the anti-programmed cell death protein 1 (anti-PD-1) antibody gene, and this construct was co-transfected with the CAR construct into T cells. In vitro or in vivo, CAR T cells showed increased secretion of anti-PD-1 antibody under control of the chimeric promoter CIFT. pS-CIFT-αPD-1/CAR T also had similar or lower PD-1 expression, higher levels of T cell activation, more release of IFN-γ, and better antitumor activity specifically against mesothelin-positive and PD-1 ligand 1 (PD-L1)-positive cell lines. The chimeric promoter may be a promising strategy to manipulate the content of immune checkpoint inhibitors or other proteins in future therapeutic approaches for cancer treatment.

Eomesodermin promotes interaction of RelA and NFATc2 with the Ifng promoter and multiple conserved noncoding sequences across the Ifng locus in mouse lymphoma BW5147 cells

The T-box transcription factor Eomesodermin (Eomes) regulates the lineage-dependent expression of interferon γ (IFN-γ). We previously showed that Eomes promotes IFN-γ production and interacts with multiple conserved noncoding sequences (CNS) across the Ifng locus in mouse lymphoma BW5147 cells. In the present study, we investigated the transcriptional regulation of IFN-γ by the nuclear factor κB (NF-κB) subunit RelA and nuclear factor of activated T cells c2 (NFATc2, also known as NFAT1) in Eomes-transfected BW5147 cells. Eomes promoted the interaction of RelA and NFATc2 with the Ifng promoter and five CNS, including CNS-22 and CNS+30 upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). The dual NF-κB and STAT3 inhibitor TPCA-1 moderately reduced the PMA- and IM-induced IFN-γ transcription in Eomes-transfected BW5147 cells. TPCA-1 interfered with RelA binding to the Ifng promoter, CNS-22 and CNS+30. Moreover, TPCA-1 reduced the interaction of Eomes or NFATc2 with the Ifng promoter and CNS+30. The present results indicate that Eomes promotes the interaction of RelA and NFATc2 with the Ifng promoter and multiple CNS across the Ifng locus in BW5147 cells.

A New Method for Screening Natural Products to Stimulate IFN-γ Production in Jurkat Human T Lymphocytes.

Interferon-γ (IFN-γ) is a critical cytokine in the defense against viral and bacterial infection. It is mainly produced by natural killer cells and activated T cells. Given its regulatory role in coordinating cellular and humoral immune responses, IFN-γ is considered to be an effective therapeutic agent in the treatment of viral infection. Here we established a fluorescence-based high-content screening model to find small molecules that can stimulate the production of IFN-γ in human Jurkat cells. After a primary screening of 267 natural products, two hits, Astragalus polyphenols and 6-shogaol, were identified to promote the activity of the IFN-γ promoter and subsequently validated by the flow cytometry assay. Obviously, both Astragalus polyphenols and 6-shogaol exhibited potential to induce the transcription and expression of IFN-γ in a dose-dependent manner. These results indicated that our high-content screening model could be a credible and useful platform to contribute to the discovery of novel molecules to promote the expression of IFN-γ and provide leading compounds for the treatment of viral infectious diseases.

Differential interferon-γ production by naive and memory-like CD8 Tcells.

CD8 Tcells play a crucial role in immune responses to virus infections and tumors. Naïve CD8 T lymphocytes after TCR stimulation undergo differentiation into CTLs and memory cells, which are essential sources of IFN-γ. We investigated IFN-γ production by CD8 Tcell subsets found in nonimmune mice. A minor fraction of in vitro TCR-stimulated CD8 Tcells produce IFN-γ, and it is regulated at the transcriptional level. Antigen inexperienced C57BL/6 mice present the coexistence of 2 populations. The main population exhibits a CD44low CD122low profile, which is compatible with naïve lymphocytes. The minor expresses a phenotype of immunologic memory, CD44hi CD122hi . Both subsets are able to produce IL-2 in response to TCR activation, but only the memory-like population is responsible for IFN-γ production. Similar to memory CD8 Tcells, CD44hi CD8+ Tcells also present a higher level of the transcriptional factor Eomes and a lower level of T-bet (Tbx21) mRNA than CD44low CD8+ Tcells. The presence of the CD44hi CD8+ Tcell population in nonimmune OT-I transgenic mice reveals that the population is generated independently of antigenic stimulation. CpG methylation is an efficient epigenetic mechanism for gene silencing. DNA methylation at posttranscriptional CpG sites in the Ifng promoter is higher in CD44low CD8+ Tcells than in CD44hi CD8+ Tcells. Thus, memory-like CD8 Tcells have a distinct epigenetic pattern in the Ifng promoter and can rapidly produce IFN-γ in response to TCR stimulation.

A novel, non-feeder-cell approach to generate large numbers of Cytokine-Induced Memory-Like NK cells for adoptive cells therapies

Abstract We have constructed two heterodimeric multi-cytokine fusions, HCW9201 and HCW9206, to support a “Kick and Expand” approach to activate and induce proliferation of purified NK cells for adoptive cell therapy (ACT). HCW9201 is a heterodimeric fusion protein complex comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-18 and IL-12. HCW9206 is heterodimeric fusion protein comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-7, and IL-21. When purified human NK cells from peripheral blood were activated with HCW9201 for three hours and then incubated with HCW9206 in combination with a capture antibody, the HCW9201-activated NK cells expanded approximately 100–300 folds within 14 days. The expanded NK cells exhibit a cytokine-induced memory-like (CIML) phenotype with a high metabolic rate and respiratory capacity, remarkable anti-tumor activity, and persistence when they were adoptively transferred into NSG mice. They also retained their heightened responsiveness when re-stimulated with tumor targets. Cytokine dependent epigenetic demethylation imprints of the Ifng promoter region were also observed for at least 10 days after the NK cells were adoptively transferred to NSG mice. In conclusion, a simple, scalable, non-feeder-cell-based “Kick and Expand” process was developed to support the generation of large numbers of CIML NK cells for multiple rounds of ACT using peripheral blood NK cell. We also provide data to show that these CIML NK cells are an excellent source for generation of CAR-NK cells.

Functional analysis of Epinephelus coioides peroxisome proliferative–activated receptor α (PPARα): Involvement in response to viral infection

Peroxisome proliferative–activated receptor α (PPARα) belongs to the superfamily of nuclear receptors (NR). Studies have demonstrated that PPARα functions in energy metabolism, hepatic function, immune response, cell cycle, and apoptosis. In teleost fish, few studies have investigated the role of PPARα in the immune response. In this study, the grouper PPARα gene (EcPPARα) was investigated for its role in viral infection. The open reading frame of EcPPARα encoded a protein of 469 amino acids and contained an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Phylogenetic analysis revealed that EcPPARα was most closely related to homologous genes in Sander lucioperca and Perca flavescens. Upon challenge with SGIV (Singapore grouper iridovirus) and RGNNV (Red-spotted grouper nervous necrosis virus), EcPPARα expression levels were significantly upregulated in different tissues. Subcellular localization analysis showed that the EcPPARα protein localized throughout the cytoplasm and nucleus with diffuse intracellular expression patterns, which is consistent with the localization pattern of mammalian PPARs. Based on morphological observation of cytopathic effect (CPEs), viral gene expression mRNAs, and virus titer assays, the results presented here showed that an overexpression of EcPPARα promoted SGIV production in grouper spleen cells. Overexpression of EcPPARα significantly inhibited the expression of several cytokines, including interferon-related genes (IFN-γ, ISG15, MXI, MXII, MAVS and MDA5), inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and Toll like receptor adaptors (TRAF6 and MyD88). Luciferase activity of IFN-α, IFN-γ, ISRE and NF-κB promoters was also significantly decreased in EcPPARα overexpression cells. Due to these detected interferon-related genes and inflammatory cytokines play important antiviral effect against SGIV in grouper, we speculated that the promotion effect of EcPPARα on SGIV replication may be caused by down-regulation of interferon and inflammatory response. In addition, through apoptotic body observation, capspase-3 activity detection, and flow cytometry analysis, it was found that overexpression of EcPPARα promoted SGIV-induced apoptosis in fathead minnow (FHM) cells. These data may increase an understanding of the role of PPARα in fish antiviral immune responses and apoptosis.

An international validation study of the IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on T cells and a proposal for reference data for immunotoxic chemicals

SUMMARYTo evaluate the immunotoxic effects of xenobiotics, we have established the Multi-ImmunoTox assay, in which three stable reporter cell lines are used to evaluate the effects of chemicals on the IL-2, IFN-γ, IL-1β and IL-8 promoters. Here, we report the official validation study of the IL-2 luciferase assay (IL-2 Luc assay). In the Phase I study that evaluated five coded chemicals in three sets of experiments, the average within-laboratory reproducibility was 86.7%. In the Phase II study, 20 coded chemicals were evaluated at multiple laboratories. In the combined results of the Phase I and II studies, the between-laboratory reproducibility was 80.0%. These results suggested that the IL-2 Luc assay was reproducible both between and within laboratories. To determine the predictivity, we collected immunotoxicological information and constructed the reference data by classifying the chemical into immunotoxic compounds targeting T cells or others according to previously reported criteria. When compared with the reference data, the average predictivity of the Phase I and II studies was 75.0%, while that of additional 60 chemicals examined by the lead laboratory was 82.5%. Although the IL-2 Luc assay alone is not sufficient to predict immunotoxicity, it will be a useful tool when combined with other immune tests.