IFN-γ Resistance Research Articles

A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma-stimulated human cells.

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.

Dysregulation in IFN-γ signaling and response: the barricade to tumor immunotherapy.

Interferon-gamma (IFN-γ) has been identified as a crucial factor in determining the responsiveness to immunotherapy. Produced primarily by natural killer (NK) and T cells, IFN-γ promotes activation, maturation, proliferation, cytokine expression, and effector function in immune cells, while simultaneously inducing antigen presentation, growth arrest, and apoptosis in tumor cells. However, tumor cells can hijack the IFN-γ signaling pathway to mount IFN-γ resistance: rather than increasing antigenicity and succumbing to death, tumor cells acquire stemness characteristics and express immunosuppressive molecules to defend against antitumor immunity. In this review, we summarize the potential mechanisms of IFN-γ resistance occurring at two critical stages: disrupted signal transduction along the IFNG/IFNGR/JAK/STAT pathway, or preferential expression of specific interferon-stimulated genes (ISGs). Elucidating the molecular mechanisms through which tumor cells develop IFN-γ resistance help identify promising therapeutic targets to improve immunotherapy, with broad application value in conjugation with targeted, antibody or cellular therapies.

MYC Induces Immunotherapy and IFNγ Resistance Through Downregulation of JAK2.

Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

Abstract 4418: Heterogeneity of IFN-γ responsiveness in myeloid malignancies- implication for the possible impact on IFN-γ immunotherapy

Abstract Introduction: Allogeneic stem cell transplantation (alloSCT) can potentially cure hematologic malignancies through graft-versus-leukemia effect (GVL). Several studies have suggested that IFN-γ pathway is critical for GVL sensitivity or resistance in leukemia cells. In mouse model, acute myeloid leukemia (AML) generated in IFN-γ receptor-deficient mice acquired resistance to GVL (Matte-Martone, Shlomchik WD, J Clin Invest. 2017). Other groups have reported that IFN-γ restores HLA expression on myeloid blasts from patients who relapsed AMLs after alloSCT. These data support the clinical application of IFN-γ to augment GVL in subjects with post-transplant relapse. However, AML is molecularly heterogeneous, and the IFN-γ responsiveness may vary by AML subtypes. We analyzed IFN-γ responsiveness of primary AML cells and investigated the role of IFN-γ on cell death and T-cell recognition of AML cell lines. Methods: Primary AML cells collected at diagnosis (n=60), at relapse after chemotherapy (n=4), and at post-transplant relapse (n=8) were stimulated with IFN-γ at 1 ng/ml or 50 ng/mL in vitro, and IFN-γ responsiveness was analyzed by measuring phosphorylation of STAT1 (pSTAT) after 15 minutes and upregulation of HLA-DR after 48 hours by flow cytometry. U937 and THP-1 cell lines were used to investigate IFN-γ induced cell death and the impact of IFN-γ priming of leukemia cells on the activation of alloreactive T cells. For this, Jurkat cells transduced with anti-HA1 T cell receptor (αHA1-TCR-T) were used, and the activation was analyzed through CD69 upregulation by flow cytometry. Results: IFN-γ responsiveness was heterogeneous in both pSTAT1 and HLA-DR upregulation of primary AML blasts. IFN-γ responsiveness also varied within the subpopulation of leukemic blasts within the sample at diagnosis and relapse, implying clonal evolution. Compared to THP-1, U937 cells fail to upregulate HLA-DR with IFN-γ despite appropriate pSTAT1 response to IFN-γ, suggesting silencing of downstream IFN-γ activation site (GAS) and transcription of IFN-stimulated genes (ISGs). IFN-γ directly induced cell death in THP-1 in vitro, while U937 was resistant to IFN-γ induced cell death. Priming of THP-1 with IFN-γ significantly improved the affinity of αHA1-TCR-T to peptide-stimulated THP-1. Conclusion: IFN-γ responsiveness of AML blasts was heterogeneous, and cell line data suggest an impact of IFN-γ sensitivity on IFN-γ mediated cell death and alloreactive T cell recognition. Future studies aim to understand the mechanisms underlying IFN-γ sensitivity or resistance in AML, which would help to design clinical trials by selecting the patients who would obtain maximal benefit from IFN-γ therapy in post-transplant relapse (NCT04628338). Citation Format: Kedwin Ventura, Iuliia Kovalenko, Jui-En Ray Lee, Biswas Neupane, Daniel Stapor, Sawa Ito. Heterogeneity of IFN-γ responsiveness in myeloid malignancies- implication for the possible impact on IFN-γ immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4418.

Cancer Cell Resistance to IFNγ Can Occur via Enhanced Double-Strand Break Repair Pathway Activity.

The pleiotropic cytokine interferon-gamma (IFNγ) is associated with cytostatic, antiproliferation, and proapoptotic functions in cancer cells. However, resistance to IFNγ occurs in many cancer cells, and the underlying mechanism is not fully understood. To investigate potential IFNγ-resistance mechanisms, we performed IFNγ-sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ-sensitive and IFNγ-resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. Suppression of single-strand break (SSB) repair genes increased the dependency on DSB repair genes after IFNγ treatment. Furthermore, inhibition of the DSB repair pathway exhibited a synergistic effect with IFNγ treatment both in vitro and in vivo. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides comprehensive resources and evidence to elucidate a mechanism of IFNγ resistance in cancer and has the potential to inform combination therapies to overcome immunotherapy resistance.

N-glycosylation Regulates Intrinsic IFN-γ Resistance in Colorectal Cancer: Implications for Immunotherapy

Background & AimsAdvanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored. MethodsSpontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines. ResultsThe conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N-glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N-acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ–resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N-glycosylation, as well as IFNγRα protein stability and signaling. ConclusionsTogether, our results demonstrated that tumor-associated changes in N-glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC.

Upregulation of HOXC9 generates interferon-gamma resistance in gastric cancer by inhibiting the DAPK1/RIG1/STAT1 axis.

Clinical reports indicate that gastric cancer (GC) has a high mortality rate, but its pathological mechanism remains poorly understood. This work integrated bioinformatics analysis with experimental verification to explore novel biomarkers of gastric cancer. First, weighted gene coexpression network analysis was applied to screen significant genes correlated with GC development. Gene set enrichment analysis was also used to unearth the most relevant biological functions of significant genes. As a result, we discovered homeobox C9 (HOXC9) as a novel oncogene in GC, primarily through negatively regulating immune response. High expression of HOXC9 predicted a poor prognosis in GC patients, and knocking down HOXC9 efficiently enhanced the interferon‐gamma (IFNγ)‐dependent apoptosis in two GC cell lines as well as organoids from patients. Furthermore, cleaved caspase‐3/7 and phosphorylated signal transducer and activator of transcription 1 (p‐STAT1) were also significantly enhanced in HOXC9 knockdown cells and organoids treated with IFNγ. Mechanistically, we found that HOXC9 inhibited the death‐associated protein kinase 1 (DAPK1) and its downstream retinoic acid‐inducible gene‐I (RIG1) to generate GC IFNγ resistance. In summary, we identified and confirmed that HOXC9 generates IFNγ resistance in GC by inhibiting the DAPK1/RIG1/p‐STAT1 axis.

TGF-beta-mediated lung fibroblast-to-myofibroblast transition exhibits IFN-γ resistance via SHP2/SOCS3 activation

Abstract Idiopathic pulmonary fibrosis is a lethal disease of unknown etiology. Abnormal activation of TGF-β is a common cause in developing fibrotic lesions, where Src homology-2 domain-containing phosphatase 2 (SHP2) plays a critical regulator in fibroblast activation and fibrosis. We found that a marked expression of phospho-SHP2, phospho-STAT3 and the suppressor of cytokine signaling 3 (SOCS3) were detected in bleomycin-induced rat lung fibrosis, which was highly association with pulmonary parenchymal lesions and collagen deposition. Human pulmonary fibroblasts differentiated into myofibroblasts via TGF-β1 treatment were characterized with the increased bundles of F-actin, enhanced expression of α-smooth muscle actin (α-SMA), fibronectin and collagen, which also exhibited the activation of SHP2, SOCS1/3, PIAS1, STAT3, IL-6 and IL-10. An AKT-regulated glycogen synthase kinase (GSK)-3β/SHP2 axis was induced in ROS-dependent regulation during myofibroblast differentiation. The significant retardation of interferon (IFN)-γ signaling in myofibroblasts was revealed by the decrease expression of phospho-STAT1 and IFN-γ-associated genes along with IFN-γ-inducible protein 10 (IP-10) reduction. Treatment of nintedanib (bibf1120) in myofibroblasts effectively inhibits both SHP2 and SOCS3 activation accompanying by a suppression of fibroblast-to-myofibroblast transition (FMT). Moreover, synergistic treatment of nintedanib and IFN-γ markedly ameliorated TGF-β1-mediated FMT. Accordingly, we speculate that the activation of both ROS/AKT/GSK-3β/SHP2 and IL-6/STAT3/SOCS3 axis signaling causes myofibroblasts hyposensitivity to IFN-γ whereby escaping from IFN-γ immune-surveillance during FMT.

S892 INTESTINAL STEM CELL DAMAGE IN GRAFT‐VERSUS‐HOST DISEASE IS PRIMARILY CAUSED BY T‐CELL DERIVED INTERFERON‐GAMMA

Background:Graft‐versus‐host disease (GVHD) after allogeneic bone marrow transplantation (allo‐BMT) is a cause of profound clinical gastro‐intestinal disease, which is associated with severe intestinal crypt damage. Although loss of the Lgr5+ intestinal stem cell (ISC) compartment is an established component of GVHD, the mechanisms for this immune mediated damage are unknown.Aims:To examine the specific cellular interactions and molecular mechanisms underlying ISC loss in GVHD intestinal damage.Methods:We used clinically relevant major (B6 into BDF1) and minor (LP into B6) mismatched experimental allo‐BMT models to evaluate the ISC compartment after immune‐mediated damage. Crypts were harvested early post‐BMT to assess intestinal organoid forming potential as read‐out for ISC function ex vivo. To investigate possible damage mechanisms we established both mouse and human co‐culture systems of activated T‐cells with intestinal organoids and used both neutralizing antibodies and knock‐out T‐cells. Several mouse models (Lgr5‐lacZ, lgr5‐gfp‐ires‐CreERT2, Ifngr −/− , Infg −/− ) were used in in vivo and ex vivo experiments to validate results.Results:We found that Lgr5+ ISCs are rapidly depleted after pre‐transplant conditioning, but recover in recipients of minor mismatched T‐cell‐depleted (TCD) BMT by day 10 post‐BMT. In GVHD mice ISCs failed to recover. In line with these results, crypts from TCD recipients were able to form organoids ex vivo by day 10 post‐BMT, while crypts from GVHD mice remained impaired, indicating immune‐mediated injury to the ISC compartment (Fig. 1A). Activated allo‐T‐cells significantly reduced ISC function ex vivo in both human and mouse co‐culture systems as evidenced by reduced organoid growth (Fig. 1B). Screening numerous effector mechanisms, we found that IFNγ blockade significantly protected organoid formation (Fig. 1C), confirmed by IFNγ resistance of IFNγ receptor knockout (IFNγR −/− ) organoids. Furthermore, culture of mouse or human organoids with IFNγ in the absence of T‐cells indicated that IFNγ was sufficient for organoid suppression. We tested if ISC damage is indirectly caused by damage to its supporting Paneth cell (PC) niche. However, using organoid cultures from PC deficient mice and from PC and/or ISC‐specific IFNγR −/− KO mice, we determined it is the ISCs themselves that are sensitive to IFNγ toxicity. Ruxolitinib, a selective JAK1/2 inhibitor in clinical investigation, protected intestinal organoids from allo‐T‐cells as well as from direct IFNγ toxicity (Fig. 1D). We validated our findings in vivo and found that both anti‐IFNγ and Ruxolitinib treatment protected ISC from elimination on day 10 post‐BMT (Fig. 1E). When using IFNγ −/− T‐cells in allo‐BMT to eliminate T‐cell‐derived IFNγ effects, we found significantly less damage to the ISC compartment than in the GvHD control with WT T‐cells (Fig. 1F). Gene expression analysis revealed the initiation of an apoptotic program in ISCs upon IFNγ exposure, with decreased anti‐apoptotic Bcl2 and Bcl2l1 and increased apoptotic Bak1 expression. In addition, we found increased apoptosis by Annexin V analysis and activity and presence of cleaved caspase 3 after IFNγ stimulation.Summary/Conclusion:Our findings reveal that damage to the ISC compartment is a common feature of GvHD, and T‐cell derived IFNγ plays a central role in ISC damage and elimination, instituting a gene expression program resulting in ISC apoptosis. Jak/STAT signaling inhibition can protect target tissue stem cells and as such may provide a promising approach for protecting epithelial tissues from T‐cell mediated damage in GVHD.image

Serum Interleukin Levels and Insulin Resistance in Major Depressive Disorder.

Major depressive disorder (MDD) has been associated with inflammatory processes, including increased cytokine levels, even in individuals who are otherwise physically healthy, while some MDD patients may show insulin resistance (IR). However, correlations between cytokines and IR parameters have not been studied extensively in MDD. In the present study, we measured IL-1β, IL-4, IFN-γ, TGF-β1, insulin and glucose in 63 MDD patients and 27 healthy controls. The associations between cytokine levels and IR were examined. The results revealed a significant increase (p<0.05) in serum levels of IL-1β, IL-4, IFN-γ, TGF-β1, insulin, insulin/glucose ratio, and insulin resistance (HOMA2IR) in MDD patients as compared with controls. There was a significant correlation between HOMA2IR with both IFN-γ (ρ=0.289, p<0.05) and TGF-β1 (ρ=0.364, p<0.05). The present study further confirms that MDD is accompanied by activation of the immune system with significant elevations in the levels of four cytokines. These results indicate stimulation of the immune system and increased IR and modulation of IR by increased cytokine levels in MDD. These findings show that immune activation and associated IR are a new drug target in depression.

Silencing of the interferon-inducible gene Ifi204/p204 induces resistance to interferon-γ-mediated cell growth arrest of tumor cells

Many tumor cells escape from cancer immunosurveillance and resist treatment with interferons (IFNs). Although the mechanism underlying IFN resistance is mostly attributed to a deficiency of components of the IFN-signaling pathway, some types of tumor cells resist IFN-mediated cell growth arrest despite the presence of an intact JAK/STAT signaling pathway. However, the molecular mechanisms underlying the unresponsiveness to IFNs independent of the defective JAK/STAT pathway remain to be clarified. To elucidate the mechanisms underlying IFNγ resistance, we examined the anti-proliferative effect of IFNγ on mouse tumor cell lines. Mouse squamous cell carcinoma (SCCVII) cells were resistant to IFNγ-mediated cell growth arrest despite the presence of the IFNγ-induced STAT1-dependent signaling pathway, whereas IFNγ inhibited cell growth of B16/F1 cells, a well-known IFNγ-sensitive mouse melanoma cell line, at the G1 phase of the cell cycle. Treatment of SCCVII cells with IFNγ neither downregulated the expression of cyclin D1, cyclin A2, and cyclin E1 nor induced a hypo-phosphorylated, active form of retinoblastoma protein (pRb). Interestingly, the hyper-phosphorylated, inactive form of pRb was exclusively localized in the cytoplasm in SCCVII cells. The IFN-inducible 204 gene (Ifi204), whose gene product, p204, binds to pRb and exerts an anti-proliferative effect, was repressed in SCCVII cells. p204 overexpression in SCCVII significantly inhibited cell growth, and mutation of a pRb-binding LXCXE motif decreased the anti-proliferative effect. These results suggest that silencing of Ifi204/p204 induces resistance to IFNγ-mediated cell growth arrest in SCCVII cells.

Tumor CDKN2A-Associated JAK2 Loss and Susceptibility to Immunotherapy Resistance

Poor clinical responses to checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies in melanoma have recently been associated with acquired IFNγ resistance that protects tumor cells from the antiproliferative and pro-apoptotic cytokine activity. IFNγ-resistant melanoma cells very often lack functional expression of the IFNγ signaling pathway gene JAK2 due to gene deletions or inactivating gene mutations. Analyzing melanoma cell lines (n = 46, applying next-generation targeted sequencing and single nucleotide polymorphism arrays) as well as available genomic data sets from The Cancer Genome Atlas (TCGA) tumor tissue samples (cutaneous melanoma n = 367, lung squamous cell carcinoma n = 501, bladder urothelial carcinoma n = 408, breast invasive carcinoma n = 768, colorectal adenocarcinoma n = 257), we demonstrate that the frequent chromosomal losses of the tumor suppressor CDKN2A in melanoma and other tumor entities enhance the susceptibility to IFNγ resistance by concomitant deletion of the JAK2 gene (odds ratio = 223.17, 95% confidence interval = 66.91 to 1487.38, two-sided P = 7.6×10-46). Tumors with JAK2 mutations or homozygous JAK2 deletions demonstrate allelic losses covering both CDKN2A and JAK2. This suggests that patients with tumor chromosomal CDKN2A losses are susceptible to developing immunotherapy resistance and should be screened for JAK2 deficiency prior to and under immune checkpoint blocking therapy.

Acquired IFN\u03b3 resistance impairs anti-tumor immunity and gives rise to T-cell-resistant melanoma lesions

Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.

An increase in galectin-3 causes cellular unresponsiveness to IFN-γ-induced signal transduction and growth inhibition in gastric cancer cells.

Glycogen synthase kinase (GSK)-3β facilitates interferon (IFN)-γ signaling by inhibiting Src homology-2 domain-containing phosphatase (SHP) 2. Mutated phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN) cause AKT activation and GSK-3β inactivation to induce SHP2-activated cellular unresponsiveness to IFN-γ in human gastric cancer AGS cells. This study investigated the potential role of galectin-3, which acts upstream of AKT/GSK-3β/SHP2, in gastric cancer cells. Increasing or decreasing galectin-3 altered IFN-γ signaling. Following cisplatin-induced galectin-3 upregulation, surviving cells showed cellular unresponsiveness to IFN-γ. Galectin-3 induced IFN-γ resistance independent of its extracellular β-galactoside-binding activity. Galectin-3 expression was not regulated by PI3K activation or by a decrease in PTEN. Increased galectin-3 may cause GSK-3β inactivation and SHP2 activation by promoting PDK1-induced AKT phosphorylation at a threonine residue. Overexpression of AKT, inactive GSK-3βR96A, SHP2, or active SHP2D61A caused cellular unresponsiveness to IFN-γ in IFN-γ-sensitive MKN45 cells. IFN-γ-induced growth inhibition and apoptosis in AGS cells were observed until galectin-3 expression was downregulated. These results demonstrate that an increase in galectin-3 facilitates AKT/GSK-3β/SHP2 signaling, causing cellular unresponsiveness to IFN-γ.

Polycomb Repressive Complex 2 Confers BRG1 Dependency on the CIITA Locus.

CIITA (or MHC2TA) coordinates constitutive and IFN-γ-induced expression of MHC class II genes. IFN-γ responsiveness of CIITA requires BRG1 (SMARCA4), the ATPase engine of the chromatin remodeling SWI/SNF complex (also called BAF). SWI/SNF is defective in many human cancers, providing a mechanism to explain IFN-γ resistance. BRG1 dependency is mediated through remote elements. Short CIITA reporters lacking these elements respond to IFN-γ, even in BRG1-deficient cells, suggesting that BRG1 counters a remote repressive influence. The nature of this distal repressor is unknown, but it would represent a valuable therapeutic target to reactivate IFN-γ responsiveness in cancer. In this article, we show that the polycomb repressive complex 2 (PRC2) components EZH2 and SUZ12, as well as the associated histone mark H3K27me3, are codetected at interenhancer regions across the CIITA locus. IFN-γ caused a BRG1-dependent reduction in H3K27me3, associated with nucleosome displacement. SUZ12 knockdown restored IFN-γ responsiveness in BRG1-null cells, and it mimicked the ability of BRG1 to induce active histone modifications (H3K27ac, H3K4me) at the -50-kb enhancer. Thus, PRC2 confers BRG1 dependency on the CIITA locus. Our data suggest that, in addition to its known roles in promoting stemness and proliferation, PRC2 may inhibit immune surveillance, and it could be targeted to reactivate CIITA expression in SWI/SNF deficient cancers.

Mutational Analysis of the Chlamydia muridarum Plasticity Zone.

Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. Chlamydia trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed--and some may produce functional proteins--but are dispensable for infection of the murine genital tract.

Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of Gamma Interferon via Signaling Attenuation.

Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-γ) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN-γ in EV71 infection using a murine model, showed that IFN-γ was elevated. Moreover, IFN-γ receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice. In vitro results showed that IFN-γ pretreatment reduced EV71 yield, whereas EV71 infection caused IFN-γ resistance with attenuated IFN-γ signaling in IFN regulatory factor 1 (IRF1) gene transactivation. To study the immunoediting ability of EV71 proteins in IFN-γ signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN-γ-induced IRF1 transactivation following a loss of signal transducer and activator of transcription 1 (STAT1) nuclear translocation. Viral 3D attenuated IFN-γ signaling accompanied by a STAT1 decrease without interfering with IFN-γ receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN-γ signaling. Interestingly, viral 2A attenuated IFN-γ signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN-γ and the immunoediting ability by EV71 2A and 3D, which attenuate IFN-γ signaling through different mechanisms. Immunosurveillance by gamma interferon (IFN-γ) may confer anti-enterovirus 71 (anti-EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. IFN-γ receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-γ. However, EV71 infection caused cellular insusceptibility in response to IFN-γ stimulation. We used an in vitro system with viral protein expression to explore the novel IFN-γ inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-γ, particularly for IFN-γ-mediated anti-EV71 activity.

Helicobacter pylori infection activates Src homology-2 domain-containing phosphatase 2 to suppress IFN-γ signaling.

Helicobacter pylori infection not only induces gastric inflammation but also increases the risk of gastric tumorigenesis. IFN-γ has antimicrobial effects; however, H. pylori infection elevates IFN-γ-mediated gastric inflammation and may suppress IFN-γ signaling as a strategy to avoid immune destruction through an as-yet-unknown mechanism. This study was aimed at investigating the mechanism of H. pylori-induced IFN-γ resistance. Postinfection of viable H. pylori decreased IFN-γ-activated signal transducers and activators of transcription 1 and IFN regulatory factor 1 not only in human gastric epithelial MKN45 and AZ-521 but also in human monocytic U937 cells. H. pylori caused an increase in the C-terminal tyrosine phosphorylation of Src homology-2 domain-containing phosphatase (SHP) 2. Pharmacologically and genetically inhibiting SHP2 reversed H. pylori-induced IFN-γ resistance. In contrast to a clinically isolated H. pylori strain HP238, the cytotoxin-associated gene A (CagA) isogenic mutant strain HP238(CagAm) failed to induce IFN-γ resistance, indicating that CagA regulates this effect. Notably, HP238 and HP238(CagAm) differently caused SHP2 phosphorylation; however, imaging and biochemical analyses demonstrated CagA-mediated membrane-associated binding with phosphorylated SHP2. CagA-independent generation of reactive oxygen species (ROS) contributed to H. pylori-induced SHP2 phosphorylation; however, ROS/SHP2 mediated IFN-γ resistance in a CagA-regulated manner. This finding not only provides an alternative mechanism for how CagA and ROS coregulate SHP2 activation but may also explain their roles in H. pylori-induced IFN-γ resistance.

Autophagy facilitates cytokine-induced ICAM-1 expression

ICAM-1 can be induced by inflammatory cytokines such as IFN-γ and TNF-α. This study investigated whether autophagy regulates ICAM-1 given that autophagy facilitates signaling of these two cytokines. Exogenous IFN-γ induced ICAM-1 in human lung epithelial A549 cells carrying wild type p53, a transcription factor reported for ICAM-1, but not in PC14PE6/AS2 (AS2) cells carrying mutated p53. However, IFN-γ also induced ICAM-1 in A549 cells with short hairpin RNA-silenced p53. No changes in IFN-γ receptor expression were observed in AS2 cells, but IFN-γ-activated Jak2/STAT1/IFN regulatory factor 1 was markedly decreased. In AS2 cells, increased levels of reactive oxygen species induced the activation of Src homology domain-containing phosphatase 2 (SHP2), while SHP2 was essential for IFN-γ resistance. AS2 cells showed autophagy resistance, and the manipulation of the autophagy pathway altered IFN-γ resistance. Aberrant Bcl-2 expression and mammalian target of rapamycin activation contributed to both autophagy resistance and IFN-γ resistance. Autophagy, but not p53, also modulated TNF-α-induced NF-κB activation and ICAM-1 expression. Inhibiting autophagy decreased the adhesion of human monocytic U937 cells to IFN-γ-treated A549 cells. These results demonstrated that IFN-γ and TNF-α induced ICAM-1 expression through a common pathway that was regulated by autophagy, but not p53.

Regulation of SHP2 by PTEN/AKT/GSK-3β signaling facilitates IFN-γ resistance in hyperproliferating gastric cancer (162.8)

Abstract In addition to oncogenic activation, and tumor suppressor gene inactivation escape from immune surveillance, such as IFN-γ resistance, is critical for cancer cell growth and survival. In this study, we investigated the crosstalk signaling between IFN-γ resistance and signaling of hyperproliferation in gastric cancer cells. IFN-γ inhibited the cell growth of MKN45 cells but not hyperproliferating AGS cells. AGS cells did not respond to IFN-γ because of a decrease in STAT1 but not due to dysfunctional IFN-γ receptors. Signaling of PI3K/AKT, as well as MEK/ERK, was required for the hyperproliferation; notably, PI3K/AKT alone mediated the IFN-γ resistance. Aberrant Src homology-2 domain-containing phosphatase (SHP) 2 determined IFN-γ resistance but unexpectedly had no effects on hyperproliferation or ERK activation. In the IFN-γ resistant cells, inactivation of glycogen synthase kinase (GSK)-3β by PI3K/AKT was important for SHP2 activation but not for hyperproliferation. An imbalance of AKT/GSK-3β/SHP2 caused by a reduction of PTEN was important for the crosstalk between IFN-γ resistance and hyperproliferation. PI3K is constitutively expressed in AGS cells and immunohistochemical staining showed a correlation between hyperproliferation and expression of SHP2 and STAT1 in gastric tumors. These results demonstrate the effects of PTEN/AKT/GSK-3β/SHP2 signaling on IFN-γ resistance in hyperproliferating gastric cancer cells.