Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity.

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

RSAD2 is abundant in atherosclerotic plaques and promotes interferon-induced CXCR3-chemokines in human smooth muscle cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r = 0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice.

OBJECTIVE: Recurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models. METHODS: Exosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR. RESULTS: IFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3' UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity. CONCLUSION: Our study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal-fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.

[Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis].

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

Interferon-γ and infectious diseases: Lessons and prospects.

Infectious diseases continue to claim many lives. Prevention of morbidity and mortality from these diseases would benefit not just from new medicines and vaccines but also from a better understanding of what constitutes protective immunity. Among the major immune signals that mobilize host defense against infection is interferon-γ (IFN-γ), a protein secreted by lymphocytes. Forty years ago, IFN-γ was identified as a macrophage-activating factor, and, in recent years, there has been a resurgent interest in IFN-γ biology and its role in human defense. Here we assess the current understanding of IFN-γ, revisit its designation as an "interferon," and weigh its prospects as a therapeutic against globally pervasive microbial pathogens.

Predicting the mechanism of action of YQYYJD prescription in the treatment of non-small cell lung cancer using transcriptomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The efficacy of the herbal formula Yiqi Yangyin Jiedu (YQYYJD) in the treatment of advanced lung cancer has been reported in clinical trials. However, the key anti-lung cancer herbs and molecular mechanisms underlying its inhibition of lung cancer are not well-understood. AIM OF THE STUDY: To identify the key anti-lung cancer herbs in the YQYYJD formula and investigate their therapeutic effect and potential mechanism of action in non-small cell lung cancer (NSCLC) using transcriptomics and bioinformatics techniques. MATERIALS AND METHODS: A mouse Lewis lung carcinoma (LLC) subcutaneous inhibitory tumor model was established with 6 mice in each group. Mice were treated with the YQYYJD split formula: Yiqi Formula (YQ), Yangyin Formula (YY), and Ruanjian Jiedu Formula (RJJD) for 14 days. The tumor volume and mouse weight were recorded, and the status of tumor occurrence was further observed by taking photos. The tumor was stained with hematoxylin-eosin to observe its histopathological changes. Immunohistochemistry was used to detect the expression of the proliferation marker Ki67 and the apoptotic marker Caspase-3 in tumor tissues. Flow cytometry was used to detect the number of CD4+ and CD8+ T cells and cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in the spleen and tumor tissues. The differential genes of key drugs against tumors were obtained by transcriptome sequencing of tumors. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed on differential genes to obtain pathways and biological processes where targets were aggregated. TIMER2.0 and TISIDB databases were used to evaluate the impact of drugs on immune cell infiltration and immune-related genes. The binding activity of the key targets and compounds was verified by molecular docking. RESULTS: YQ, YY, and RJJD inhibited the growth of subcutaneous transplanted tumors in LLC mice to varying degrees and achieved antitumor effects by inhibiting the expression of tumor cell proliferation, apoptosis, and metastasis-related proteins. Among the three disassembled prescriptions, YQ better inhibited the growth of subcutaneous transplanted tumors in LLC mice, significantly promoted tumor necrosis, significantly increased the expression of Caspase-3 protein in tumor tissue, and significantly decreased the expression of Ki-67 (P < 0.05), thereby increasing the infiltration of CD8+ T cells. YQ significantly increased the expression of CD4+ and CD8+ T cells in tumor and splenic tissues of tumor-bearing mice and up-regulated the expression of IL-2 and IFN-γ. Transcriptome sequencing and bioinformatics results showed that after YQ intervention, differentially expressed genes were enriched in more than one tumor-related pathway and multiple immune regulation-related biological functions. There were 12 key immune-related target genes. CONCLUSION: YQ was the key disassembled prescription of YQYYJD, exerting significant antitumor effects and immune regulation effects on NSCLC. It may have relieved T cell exhaustion and regulated the immune microenvironment to exert antitumor effects by changing lung cancer-related targets, pathways, and biological processes.

IgG from Dermatophagoides pteronyssinus (Der p)-atopic individuals modulates non-atopic thymic B cell phenotype (alfa-4/beta-7) and cytokine production (IFN-γ, IL-9, and IL-10) with direct membrane interaction.

Studies about thymic B cells are scarce in the literature, but it was suggested that they can exert modulatory and regulatory functions on the immune system. Thymic B cells can play some role in regulating the most frequent allergic background worldwide, the atopy induced by the mite Dermatophagoides pteronyssinus (Der p). Here, we aimed to evaluate if the polyclonal IgG repertoire produced by Der p-atopic individuals can influence the homing and cytokine profile of human thymic B derived from non-atopic children aged less than seven days. With this purpose, we produced polyclonal IgG formulations and cultivated human thymocytes in their presence. We also assessed IgG subclasses and the direct interaction of IgG with thymic B cell membranes. Our results could demonstrate that Der p-atopic IgG could not reduce the expression of α4ß7 homing molecule as observed in response to the other IgG formulations and could reduce the frequency of IFN-γ- and IL-9-producing thymic B cells compared to the mock condition. Der p-atopic IgG could also induce thymic IL-10-producing B cells compared to control conditions. The IgG derived from Der p-atopic individuals failed to diminish the population of IL-13-producing thymic B cells, unlike the reduction observed with other IgG formulations when compared to the mock condition. All IgG formulations had similar levels of IgG subclasses and directly interacted with thymic B cell membranes. Finally, we performed experiments using peripheral non-atopic B cells where IgG effects were not observed. In conclusion, our observation demonstrates that IgG induced in allergic individuals can modulate non-atopic thymic B cells, potentially generating thymic B cells prone to allergy development, which seems to not occur in mature B cells.

Cycloolivil Isolated from Nardostachys jatamansi Inhibits TNF-α/IFN-γ-Induced Chemokine Production by Blocking NF-κB and JAK/STAT Activation in HaCaT Keratinocytes.

Nardostachys jatamansi is widely used as a traditional medicine in Asian countries. Numerous recent studies have reported the biological activities of its secondary metabolites and extracts. In this study, a total of 14 components were isolated, including cycloolivil and 2-(3'-hydroxy-5'-ethoxyphenyl)-3-hydroxylmethyl-7-methoxy-2,3-dihydrobenzofuran-5-carboxylic acid, which were first discovered in N. jatamansi. The isolated compounds were investigated for their anti-inflammatory effects on HaCaT keratinocytes and their potential to alleviate skin inflammation. The results of the screening revealed that cycloolivil and 4ß-hydroxy-8ß-methoxy-10-methylene-2,9-dioxatricyclo[4.3.1.03,7]decane reduced the production of inflammatory cytokines induced by TNF-α/IFN-γ, such as IL-6, IL-8, and RANTES, in keratinocytes. This study focused on exploring the biological effects of cycloolivil, and the results suggested that cycloolivil inhibits the expression of COX-2 proteins. Further mechanistic evaluations confirmed that the anti-inflammatory effects of cycloolivil were mediated by blockage of the NF-κB and JAK/STAT signaling pathways. These results suggest that cycloolivil isolated from N. jatamansi could be used to treat skin inflammatory diseases.

Characterization of tumoricidal activities mediated by a novel immune cell regimen composing interferon-producing killer dendritic cells and tumor-specific cytotoxic T lymphocytes.

BACKGROUND: Although immune cell therapy has long been used for treating solid cancer, its efficacy remains limited. Interferon (IFN)-producing killer dendritic cells (IKDCs) exhibit cytotoxicity and present antigens to relevant cells; thus, they can selectively induce tumor-associated antigen (TAA)-specific CD8 T cells and may be useful in cancer treatment. Various protocols have been used to amplify human IKDCs from peripheral sources, but the complexity of the process has prevented their widespread clinical application. Additionally, the induction of TAA-specific CD8 T cells through the adoptive transfer of IKDCs to immunocompromised patients with cancer may be insufficient. Therefore, we developed a method for generating an immune cell-based regimen, Phyduxon-T, comprising a human IKDC counterpart (Phyduxon) and expanded TAA-specific CD8 T cells. METHODS: Peripheral blood mononuclear cells from ovarian cancer patients were cultured with human interleukin (hIL)-15, hIL-12, and hIL-18 to generate Phyduxon-T. Then, its phenotype, cytotoxicity, and antigen-presenting function were evaluated through flow cytometry using specific monoclonal antibodies. RESULTS: Phyduxon exhibited the characteristics of both natural killer and dendritic cells. This regimen also exhibited cytotoxicity against primary ovarian cancer cells and presented TAAs, thereby inducing TAA-specific CD8 T cells, as evidenced by the expression of 4-1BB and IFN-γ. Notably, the Phyduxon-T manufacturing protocol effectively expanded IFN-γ-producing 4-1BB+ TAA-specific CD8 T cells from peripheral sources; these cells exhibited cytotoxic activities against ovarian cancer cells. CONCLUSIONS: Phyduxon-T, which is a combination of natural killer cells, dendritic cells, and TAA-specific CD8 T cells, may enhance the efficacy of cancer immunotherapy.

The central role of natural killer cells in mediating acute myocarditis after mRNA COVID-19 vaccination.

BACKGROUND: Vaccine-related acute myocarditis is recognized as a rare and specific vaccine complication following mRNA-based COVID-19 vaccinations. The precise mechanisms remain unclear. We hypothesized that natural killer (NK) cells play a central role in its pathogenesis. METHODS: Samples from 60 adolescents with vaccine-related myocarditis were analyzed, including pro-inflammatory cytokines, cardiac troponin T, genotyping, and immunophenotyping of the corresponding activation subsets of NK cells, monocytes, and T cells. Results were compared with samples from 10 vaccinated individuals without myocarditis and 10 healthy controls. FINDINGS: Phenotypically, high levels of serum cytokines pivotal for NK cells, including interleukin-1ß (IL-1ß), interferon α2 (IFN-α2), IL-12, and IFN-γ, were observed in post-vaccination patients with myocarditis, who also had high percentage of CD57+ NK cells in blood, which in turn correlated positively with elevated levels of cardiac troponin T. Abundance of the CD57+ NK subset was particularly prominent in males and in those after the second dose of vaccination. Genotypically, killer cell immunoglobulin-like receptor (KIR) KIR2DL5B(-)/KIR2DS3(+)/KIR2DS5(-)/KIR2DS4del(+) was a risk haplotype, in addition to single-nucleotide polymorphisms related to the NK cell-specific expression quantitative trait loci DNAM-1 and FuT11, which also correlated with cardiac troponin T levels in post-vaccination patients with myocarditis. CONCLUSION: Collectively, these data suggest that NK cell activation by mRNA COVID-19 vaccine contributed to the pathogenesis of acute myocarditis in genetically and epidemiologically vulnerable subjects. FUNDING: This work was funded by the Hong Kong Collaborative Research Fund (CRF) 2020/21 and the CRF Coronavirus and Novel Infectious Diseases Research Exercises (reference no. C7149-20G).

Melatonin improves salivary gland damage and hypofunction in pSS by inhibiting IL-6/STAT3 signaling through its receptor-dependent manner.

OBJECTIVE: Primary Sjogren's syndrome (pSS) is an autoimmune disease of the exocrine glands with no specific or efficient treatments. Melatonin, a natural hormone, is revealed to show multiple biological functions, both receptor-dependent and independent effects, including anti-apoptotic, antioxidant, and anti-inflammatory activities. However, the potential mechanism by which melatonin protects salivary glands (SGs) of pSS from damage needs to be clarified. The purpose of current study was to explore the role and receptor-related mechanisms of melatonin in pSS-induced glandular damage. METHODS AND RESULTS: NOD/Ltj mice were used to spontaneously mimic pSS-induced glandular hypofunction in vivo and primary human salivary gland epithelial (HSGE) cells were stimulated by interferon-γ (IFN-γ) to mimic pSS-induced inflammation in SGs cells in vitro. Melatonin-treated mice exhibited a significant reduction in SG injury of NOD/Ltj mice, which was accompanied by an increase in salivary flow rate, a decrease in inflammatory infiltration within the gland, and a suppression of oxidative stress indicators as well as cell apoptosis. Notably, both melatonin membrane receptors and nuclear receptors played an important role in the anti-apoptotic effects of melatonin on the SGs of NOD/Ltj mice. Furthermore, melatonin blocked the IL-6/STAT3 pathway through receptor-dependent manners in IFN-γ-stimulated HSGE cells. However, it was evident that the anti-oxidative and anti-apoptotic properties of melatonin on IFN-γ-stimulated HSGE cells were diminished by IL-6 treatment. CONCLUSION: Melatonin had the potential to mitigate inflammation, oxidative stress, and apoptosis in SGs of pSS by inhibiting the IL-6/STAT3 pathway through receptor-dependent mechanisms. This intervention effectively prevented glandular damage and preserved functional integrity.

IFNγ insufficiency during mouse intra-vaginal Chlamydia trachomatis infection exacerbates alternative activation in macrophages with compromised CD40 functions.

Chlamydia trachomatis (C.tr), an obligate intracellular pathogen, causes asymptomatic genital infections in women and is a leading cause of preventable blindness. We have developed in vivo mouse models of acute and chronic C. trachomatis genital infection to explore the significance of macrophage-directed response in mediating immune activation/suppression. Our findings reveal that during chronic and repeated C. trachomatis infections, Th1 response is abated while Treg response is enhanced. Additionally, an increase in exhaustion (PD1, CTLA4) and anergic (Klrg3, Tim3) T cell markers is observed during chronic infection. We have also observed that M2 macrophages with low CD40 expression promote Th2 and Treg differentiation leading to sustained C. trachomatis genital infection. Macrophages infected with C. trachomatis or treated with supernatant of infected epithelial cells drive them to an M2 phenotype. C. trachomatis infection prevents the increase in CD40 expression as observed in western blots and flow cytometric analysis. Insufficient IFNγ, as observed during chronic infection, leads to incomplete clearance of bacteria and poor immune activation. C. trachomatis decapacitates IFNγ responsiveness in macrophages via hampering IFNγRI and IFNγRII expression which can be correlated with poor expression of MHC-II, CD40, iNOS and NO release even following IFNγ supplementation. M2 macrophages during C. trachomatis infection express low CD40 rendering immunosuppressive, Th2 and Treg differentiation which could not be reverted even by IFNγ supplementation. The alternative macrophages also harbour high bacterial load and are poor responders to IFNγ, thus promoting immunosuppression. In summary, C. trachomatis modulates the innate immune cells, attenuating the anti-chlamydial functions of T cells in a manner that involves decreased CD40 expression on macrophages.

Mefloquine enhances the efficacy of anti-PD-1 immunotherapy via IFN-γ-STAT1-IRF1-LPCAT3-induced ferroptosis in tumors.

BACKGROUND: Ferroptosis plays an important role in enhancing the efficacy of anti-programmed cell death 1 (PD-1) immunotherapy; however, the molecular mechanisms by which tumor ferroptosis sensitizes melanoma and lung cancer to anti-PD-1 immunotherapy have not been elucidated. METHODS: Cytotoxicity assays, colony formation assays, flow cytometry and animal experiments were used to evaluate the effects of mefloquine (Mef) on survival and ferroptosis in melanoma and lung cancer. RNA sequencing, Real-time quantitative PCR (qRT-PCR), western blotting, chromatin immunoprecipitation-qPCR and flow cytometry were used to determine the molecular mechanisms by which Mef regulates lysophosphatidylcholine acyltransferase 3 (LPCAT3). The relationship between LPCAT3 and the efficacy of anti-PD-1 immunotherapy was verified via a clinical database and single-cell RNA sequencing (ScRNA-Seq). RESULTS: In this study, we discovered that Mef induces ferroptosis. Furthermore, treatment with Mef in combination with T-cell-derived interferon-γ (IFN-γ) enhanced tumor ferroptosis and sensitized melanoma and lung cancer cells to anti-PD-1 immunotherapy. Mechanistically, Mef upregulated the expression of LPCAT3, a key gene involved in lipid peroxidation, by activating IFN-γ-induced STAT1-IRF1 signaling, and knocking down LPCAT3 impaired the induction of ferroptosis by Mef+IFN-γ. Clinically, analysis of the transcriptome and single-cell sequencing results in patients with melanoma showed that LPCAT3 expression was significantly lower in patients with melanoma than in control individuals, and LPCAT3 expression was positively correlated with the efficacy of anti-PD-1 immunotherapy. CONCLUSIONS: In conclusion, our study demonstrated a novel mechanism by which LPCAT3 is regulated, and demonstrated that Mef is a highly promising new target that can be utilized to enhance the efficacy of anti-PD-1 immunotherapy.

Reciprocal inhibition between TP63 and STAT1 regulates anti-tumor immune response through interferon-γ signaling in squamous cancer.

Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8+ T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8+ T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8+ T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.

Early application of IFNγ mediated the persistence of HBV in an HBV mouse model.

The antiviral activity of interferon gamma (IFNγ) against hepatitis B virus (HBV) was demonstrated both in vivo and in vitro in a previous study. IFNγ can suppress HBV replication by accelerating the decay of replication-competent nucleocapsids of HBV. However, in this study, we found that the direct application of the mouse IFNγ (mIFNγ) expression plasmid to the liver of an HBV hydrodynamic injection (HI) mouse model led to the persistence of HBV, as indicated by sustained HBsAg and HBeAg levels in the serum as well as an increased percentage of the HBsAg positive mice, whereas the level of HBV DNA in the serum and the expression of HBcAg in the liver were inhibited at the early stage after HI. Meanwhile, we found that the productions of both HBcAb and HBsAb were suppressed after the application of mIFNγ. In addition, we found that HBV could be effectively inhibited in mice immunized with HBsAg expression plasmid before the application of mIFNγ. Furthermore, mIFNγ showed antiviral effect and promoted the production of HBsAb when the mice subjected to the core-null HBV plasmid. These results indicate that the application of mIFNγ in the HBV HI mouse model, the mice showed defective HBcAg-specific immunity that impeded the production of HBcAb and HBsAb, finally allowing the persistence of the virus. Moreover, IFNγ-induced negative immune regulatory factors also play an important role in virus persistence.

Nucleic Acid and Nanomaterial Synergistic Amplification Enables Dual Targets of Ultrasensitive Fluorescence Quantification to Improve the Efficacy of Clinical Tuberculosis Diagnosis.

Interferon-γ (IFN-γ) release assays (IGRAs) are constrained by the limited diagnostic performance of a single indicator and the excessive Mycobacterium tuberculosis (Mtb) antigen stimulation time. This study presents a simultaneous, homogeneous, rapid, and ultrasensitive fluorescence quantification strategy for IFN-γ and IFN-γ-induced protein 10 (IP-10). This method relies on the high-affinity binding of aptamers to IFN-γ and IP-10, the enzyme-free catalytic hairpin assembly reaction, and the heightened sensitivity of CdTe quantum dots to Ag+ and hairpin structure C-Ag+-C and carbon dots to Hg2+ and hairpin structure T-Hg2+-T. Under optimized conditions, the selectivity of IFN-γ and IP-10 was excellent, with a linear range spanning from 1 to 100 ag/mL and low limits of detection of 0.3 and 0.5 ag/mL, respectively. Clinical practicality was confirmed through testing of 57 clinical samples. The dual-indicator combination detection showed 92.8% specificity and 93.1% sensitivity, with an area under the curve of 0.899, representing an improvement over the single-indicator approach. The Mtb antigen stimulation time was reduced to 8 h for 6/7 clinical samples. These findings underscore the potential of our approach to enhance the efficiency and performance of a tuberculosis (TB) clinical diagnosis.

Intratumoral injection of interferon gamma promotes the efficacy of anti-PD1 treatment in colorectal cancer.

Immune checkpoint inhibitors (ICIs) offer new options for the treatment of patients with solid cancers worldwide. The majority of colorectal cancers (CRC) are proficient in mismatch-repair (pMMR) genes, harboring fewer tumor antigens and are insensitive to ICIs. These tumors are often found to be immune-deserted. We hypothesized that forcing immune cell infiltration into the tumor microenvironment followed by immune ignition by PD1 blockade may initiate a positive immune cycle that can boost antitumor immunity. Bioinformatics using a public database suggested that IFNγ was a key indicator of immune status and prognosis in CRC. Intratumoral administration of IFNγ increased immune cells infiltration into the tumor, but induced PD-L1 expression. A combined treatment strategy using IFNγ and anti-PD-1 antibody significantly increased T cell killing of tumor cells in vitro and showed synergistic inhibition of tumor growth in a mouse model of CRC. CyTOF found drastic changes in the immune microenvironment upon combined immunotherapy. Treatment with IFNγ and anti-PD1 antibody in CT26 tumors significantly increased infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). IFNγ had a more pronounced effect in decreasing intratumoral M2-like macrophages, while PD1 blockade increased the population of CD8+Ly6C + T cells in the tumor microenvironment, creating a more pro-inflammatory microenvironment. Additionally, PD1 induced increased expression of lymphocyte activating 3 (LAG3) in a significant fraction of CD8+ T cells and Treg cells, indicating potential drug resistance and feedback mechanisms. In conclusion, our work provides preclinical data for the Combined immunotherapy of CRC using intratumoral delivery of IFNγ and systemic anti-PD1 monoclonoal antibody.

GPR41 and GPR43 regulate CD8+ T cell priming during herpes simplex virus type 1 infection.

Naïve CD8+ T cells need to undergo a complex and coordinated differentiation program to gain the capacity to control virus infections. This not only involves the acquisition of effector functions, but also regulates the development of a subset of effector CD8+ T cells into long-lived and protective memory cells. Microbiota-derived metabolites have recently gained interest for their influence on T cells, but much remains unclear about their role in CD8+ T cell differentiation. In this study, we investigated the role of the G protein-coupled receptors (GPR)41 and GPR43 that can bind microbiota-derived short chain fatty acids (SCFAs) in CD8+ T cell priming following epicutaneous herpes simplex virus type 1 (HSV-1) infection. We found that HSV-specific CD8+ T cells in GPR41/43-deficient mice were impaired in the antigen-elicited production of interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), granzyme B and perforin, and failed to differentiate effectively into memory precursors. The defect in controlling HSV-1 at the site of infection could be restored when GPR41 and GPR43 were expressed exclusively by HSV-specific CD8+ T cells. Our findings therefore highlight roles for GPR41 and GPR43 in CD8+ T cell differentiation, emphasising the importance of metabolite sensing in fine-tuning anti-viral CD8+ T cell priming.

CD4 T cell-secreted IFN-γ in Sjögren's syndrome induces salivary gland epithelial cell ferroptosis.

BACKGROUND: Sjögren's syndrome (SS) is a chronic autoimmune disease that predominantly affects exocrine glands. Previous studies have demonstrated that upregulated interferon-gamma (IFN-γ) in SS triggers ferroptosis in salivary gland epithelial cells (SGECs), resulting in impaired salivary gland secretion. However, the immune cells responsible for secreting IFN-γ remain unclear. Therefore, this study conducted bioinformatics analysis and molecular validation to identify the origin of IFN-γ in SS salivary gland. METHODS: The 'limma' package in R software was utilized to identify differentially expressed genes (DEGs) in the human SS dataset. Subsequently, the identified DEGs were compared with the ferroptosis database and screened through Cytoscape to determine candidate genes. The cellular localization and expression patterns of candidate genes were further confirmed in the salivary gland single-cell RNA sequence (scRNA-seq) data set from healthy control and SS mice. Furthermore, in vitro and in vivo studies were performed to analyze the effect of CD4 T-secreted IFN-γ on SGECs' ferroptosis and functions. RESULTS: Upregulated TLR4, IFNG, and IL33 were screened as candidates ferroptosis ferroptosis-inducing genes in SS salivary glands. The association of IFNG and IL33 with CD4 T cells was established through immune infiltration analysis. The expression of IFN-γ on CD4 T cells was robustly higher compared with that of IL33 as evidenced by scRNA-seq and immunofluorescence co-localization. Subsequent experiments conducted on candidate genes consistently demonstrated the potent ability of IFN-γ to induce SGECs' ferroptosis and inhibit AQP5 expression. CONCLUSIONS: Our findings indicate that CD4 T cell-secreted IFN-γ in SS induces SGECs' ferroptosis and inhibits AQP5 expression.

Cellular Advanced Glycation End Products Aggravate the Immune Response in Mononuclear Cells from Patients with Type 1 Diabetes.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by immune response mediated islet beta cells destruction. However, the mechanisms that cause immune response in TIDM are still under investigation. Therefore, the goal of this study was to investigate the role of advanced glycation end products (AGEs) in the regulation of the immune response in peripheral blood mononuclear cells (PBMCs) from patients with T1DM. METHODS: PBMCs isolated from T1DM patients and control subjects were used in the current study. Cytokines, AGEs related to glyoxalase 1 (GLO1), methylglyoxal (MG)-derived AGEs were assessed longitudinally. RESULTS: The results of published T1DM PBMC microarray datasets using random-effects meta-analysis models revealed immune responses in the PBMCs of patients with T1DM compared with control subjects. Moreover, the activity of GLO1, which is the key MG-metabolizing enzyme, was significantly reduced in PBMCs from T1DM patients. We confirmed that, compared to the control subjects, GLO1 expression and activity were markedly decreased and MG-derived AGEs were significantly accumulated in the PBMCs from T1DM patients. In addition, phytohemagglutinin stimulated the secretion of tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) was positively correlated with the accumulation of cellular AGEs. Therefore, the exposure of PBMCs from control subjects to MG and a GLO1 inhibitor enhanced the accumulation of cellular MG-derived AGEs and the secretion of TNF-α and IFN-γ. CONCLUSIONS: The results of this study showed that the accumulation of cellular AGEs causes a decline in the immune response of patients with T1DM.