IFN Expression Research Articles

An Unexpected Encounter: Respiratory Syncytial Virus Nonstructural Protein 1 Interacts with Mediator Subunit MED25.

ABSTRACTHuman respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection.IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.

Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection.

Influenza virus has the ability to circumvent host innate immune system through regulating certain host factors for its effective propagation. However, the detailed mechanism is still not fully understood. Here, we report that a host sphingolipid metabolism-related factor, sphingosine kinase 2 (SPHK2), upregulated during influenza A virus (IAV) infection, promotes IAV infection in an enzymatic independent manner. The enhancement of the virus replication is not abolished in the catalytic-incompetent SPHK2 (G212E) overexpressing cells. Intriguingly, the sphingosine-1-phosphate (S1P) related factor HDAC1 also plays a crucial role in SPHK2-mediated IAV infection. We found that SPHK2 cannot facilitate IAV infection in HDAC1 deficient cells. More importantly, SPHK2 overexpression diminishes the IFN-β promoter activity upon IAV infection, resulting in the suppression of type I IFN signaling. Furthermore, ChIP-qPCR assay revealed that SPHK2 interacts with IFN-β promoter through the binding of demethylase TET3, but not with the other promoters regulated by TET3, such as TGF-β1 and IL6 promoters. The specific regulation of SPHK2 on IFN-β promoter through TET3 can in turn recruit HDAC1 to the IFN-β promoter, enhancing the deacetylation of IFN-β promoter, therefore leading to the inhibition of IFN-β transcription. These findings reveal an enzymatic independent mechanism on host SPHK2, which associates with TET3 and HDAC1 to negatively regulate type I IFN expression and thus facilitates IAV propagation.

H5N8 Subtype avian influenza virus isolated from migratory birds emerging in Eastern China possessed a high pathogenicity in mammals.

Novel H5N8 highly pathogenic avian influenza viruses (HPAIVs) bearing the clade 2.3.4.4b HA gene have been widely spread through wild migratory birds since 2020. One H5N8 HPAIV (A/Wild bird/Cixi/Cixi02/2020; here after Cixi02) was isolated from migratory birds in Zhejiang Province, Eastern China in 25 November 2020. However, its pathogenicity in avian and mammal remains unknown. Hemagglutinin gene genetic analysis indicated that Cixi02 virus belonged to the branch II of H5 clade 2.3.4.4b originated from Iraq in May 2020. Cixi02 virus showed a binding affinity to both SA α-2, 3-galactose (Gal) and SA α-2, 6 Gal receptors, good pH stability, thermostability, and replication ability in both avian and mammal cells. The poultry pathogenicity indicated that Cixi02 virus was lethal to chickens. Moreover, the mammalian pathogenicity showed that the 50% mouse lethal dose (MLD50 ) is 2.14 lgEID50 /50 μl, indicating a high pathogenicity in mice. Meanwhile, Cixi02 virus was widely detected in multiple organs, including heart, liver, spleen, lung, kidney, turbinate, and brain after nasal infection. In addition, we found high level gene expressions of TNF-α, IL-12p70, CXCL10, and IFN-α in lungs, IL-8 and IL-1β in brains, and observed severe histopathological change in lungs and brains. Collectedly, this study provided new insights on the pathogenic and zoonotic features of an H5N8 subtype AIV isolated from migratory birds.

Human endogenous retrovirus HERV-E λ 4-1 in immunopathogenesis of affective disorder

The aim of this work was to study a dependence between the production level of some pro-inflammatory cytokines by peripheral blood mononuclear cells, and activation of human endogenous retrovirus HERV-E 4-1 in the patients with recurrent depression. Patients and methods: the study included 30 patients with an verified diagnosis of recurrent depression (F 33.0) aged 26-45 years, with a disease duration of at least 3 months prior to inclusion into the study. Peripheral blood mononuclear cells were isolated by centrifugation in Ficoll density gradient (1.078 g/cm3). The human endogenous retrovirus HERV-E 4-1 env gene expression was determined by polymerase chain reaction using paired oligonucleotide primers. To assess the cytokine production, peripheral blood mononuclear cells were cultured for 24-72 hours, depending on the experimental conditions. Quantitative determination of spontaneous cytokine production was carried out by a sandwich variant of ELISA method in conditioned media from the cell cultures, according to the manufacturer instructions. Results: our data reveal higher production of IL-1 and IFN in peripheral blood mononuclear cells from those patients with recurrent depression who showed detectable HERV-E 4-1 env expression compared to the patients in whom the HERV-E 4-1 env gene expression was not detected. When studying correlation between HERV-E 4-1 env expression and production of IL-1 and IFN, a positive correlation between the studied parameters was established. Thus, taking into account our earlier data on HERV-E 4-1 immunomodulatory properties, as well as literature data concerning the HERV transcripts found in brains of mentally ill patients, along with increase of IL-1 and IFN production in the patients with recurrent depression and positive HERV-E 4-1 env gene expression, and a positive correlation between the HERV-E 4-1 env gene expression and increased level of cytokines involved in formation of pathological events in the nervous system in the patients with depression, one may conclude that activation of HERV-E 4-1 could participate in immunopathogenesis of recurrent depression by stimulating the synthesis of pro-inflammatory cytokines.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ Tcells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ Tcells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

IFIT3 and IFIT5 Play Potential Roles in Innate Immune Response of Porcine Pulmonary Microvascular Endothelial Cells to Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus.

Our previous study has demonstrated that porcine pulmonary microvascular endothelial cells (MVECs) are susceptible to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). The innate immune response of MVECs infected with HP-PRRSV would play important roles in controlling virus proliferation, resisting cellular injury, and preventing the virus from spreading to other tissues and organs. Type I interferon is one of the most effective antiviral cytokines in the innate immune response, and interferon-induced proteins with tetratricopeptide repeats (IFITs) are members of interferon-stimulated genes induced by viruses and other pathogens, which are crucial in inhibiting virus proliferation and regulating the innate immune response. However, their effects on HP-PRRSV-induced innate immunity in porcine pulmonary MVECs remain unclear. Here, the roles of IFITs in porcine pulmonary MVECs infected with the HP-PRRSV HN strain were investigated, and the effects of astragalus polysaccharides (APS), a widely used traditional Chinese herbal ingredient with the immunopotentiating effect, on them were studied. The results showed that more autophagosomes were observed in HP-PRRSV-infected MVECs, and the expression of IFN-α, IFIT3, and IFIT5 decreased or increased at different time points after infection. When silencing the genes of IFIT3 or IFIT5, the HP-PRRSV replication in MVECs was significantly increased. The expression of IFIT3 and IFIT5 could be upregulated by APS, whose inhibitory effects on the HP-PRRSV replication significantly declined when the genes of IFIT3 or IFIT5 were silenced. The results suggest that IFIT3 and IFIT5 play an important role in inhibiting the HP-PRRSV replication in porcine pulmonary MVECs, and APS suppress the multiplication of HP-PRRSV by upregulating their expression.

Sorafenib suppresses radioresistance and synergizes radiotherapy-mediated CD8+ T cell activation to eradicate hepatocellular carcinoma

Radiotherapy (RT) is applied to eradicate tumors in the clinic. However, hepatocellular carcinoma (HCC) exhibits resistance against RT. It is demonstrated that RT directly inhibits tumor growth but which induces type I interferons (IFNs) expression to phosphorylate STATs and increase STATs-downstream PD-L1 levels in the survival tumor cells. Since sorafenib is capable of suppressing STATs, we, therefore, hypothesize that sorafenib suppresses IFNs-mediated radioresistance and PD-L1 in the residual tumor cells and may synergistically enhance RT-mediated reactivation of CD8+ T immunological activity to eradicate HCC cells. We found that combined RT, sorafenib, and PBMCs significantly suppress the colony formation in the HCC cells, whereas CD8+ T cells expressed high granzyme B (GZMB) and perforin (PRF1) in co-cultured with RT-treated HCC cells. We demonstrated RT significantly inhibited HCC cell viability but induced IFNα and IL-6 expression in the RT-treated HCC cells, resulting in immune checkpoint PD-L1 and anti-apoptosis MCL1 and BCL2 overexpression in the non-RT HCC cells. We found that sorafenib decreased RT-PLC5 medium (RT-PLC5-m)-mediated cell growth by suppressing IFNα- and IL-6-mediated STAT1 and STAT3 phosphorylation. Sorafenib also reduced IFNα-mediated PD-L1 levels in HCC cells. Meanwhile, RT-PLC5-m reactivated CD8+ T cells and non-CD8+ PBMCs, resulting in high IFNγ and IL-2 levels in CD8+ T cells, and cytokines IFNα, IFNγ, IL-2, and IL-6 in non-CD8+ PBMCs. Particularly, CD8+ T cells expressed higher GZMB and PRF1 and non-CD8+ PBMCs expressed higher IFNα, IFNγ, IL-2, IL-6, CXCL9, and CXCL10 in co-cultured with RT-treated HCC cells compared to parental cells. Although we demonstrated that sorafenib slightly inhibited RT-mediated GZMB and PRF1 expression in CD8+ T cells, and cytokines levels in non-CD8+ PBMCs. Based on sorafenib significantly suppressed IFNα- and IL-6-mediated radioresistance and PD-L1 expression, we demonstrated that sorafenib synergized RT and immune surveillance for suppressing PLC5 cell viability in vitro. In conclusion, this study revealed that RT induced IFNα and IL-6 expression to phosphorylate STAT1 and STAT3 by autocrine and paracrine effect, leading to radioresistance and PD-L1 overexpression in HCC cells. Sorafenib not only suppressed IFNα- and IL-6-mediated PLC5 cell growth but also inhibited IFNα-mediated PD-L1 expression, synergistically enhancing RT-mediated CD8+ T cell reactivation against HCC cells.

ATG4B hinders porcine epidemic diarrhea virus replication through interacting with TRAF3 and activating type-I IFN signaling

Autophagy-related 4B (ATG4B) is found to exert a vital function in viral replication, although the mechanism through which ATG4B activates type-I IFN signaling to hinder viral replication remains to be explained, so far. The current work revealed that ATG4B was downregulated in porcine epidemic diarrhea virus (PEDV)-infected LLC-PK1 cells. In addition, ATG4B overexpression inhibited PEDV replication in both Vero cells and LLC-PK1 cells. On the contrary, ATG4B knockdown facilitated PEDV replication. Moreover, ATG4B was observed to hinder PEDV replication by activating type-I IFN signaling. Further detailed analysis revealed that the ATG4B protein targeted and upregulated the TRAF3 protein to induce IFN expression via the TRAF3-pTBK1-pIRF3 pathway. The above data revealed a novel mechanism underlying the ATG4B-mediated viral restriction, thereby providing novel possibilities for preventing and controlling PEDV.

Evasion of interferon-mediated immune response by arteriviruses.

IFN is the most potent antiviral cytokine required for the innate and adaptive immune responses, and its expression can help the host defend against viral infection. Arteriviruses have evolved strategies to antagonize the host cell’s innate immune responses, interfering with IFN expression by interfering with RIG, blocking PRR, obstructing IRF-3/7, NF-κB, and degrading STAT1 signaling pathways, thereby assisting viral immune evasion. Arteriviruses infect immune cells and may result in persistence in infected hosts. In this article, we reviewed the strategies used by Arteriviruses to antagonize IFN production and thwart IFN-activated antiviral signaling, mainly including structural and nonstructural proteins of Arteriviruses encoding IFN antagonists directly or indirectly to disrupt innate immunity. This review will certainly provide a better insight into the pathogenesis of the arthritis virus and provide a theoretical basis for developing more efficient vaccines.

Abstract P3077: The Role Of Neutrophils In Initiating Type I Interferon Response In Myocardial Ischemia/reperfusion

Background: Inflammation succeeding myocardial ischemia/reperfusion (MI/R) plays a significant role in damaging cardiomyocytes and influencing infarct size, with neutrophils being the most rapid and most numerous cells in the post-MI/R heart. While neutrophils play a role in wound healing, they can cause further damage through the generation of reactive oxygen species, proteolytic enzymes, and neutrophil extracellular traps (NETs). We thus hypothesize that double stranded DNA stemming from these NETs activates cGAS-STING signaling in a murine model of MI/R, leading to the production of type I interferons by neutrophils, which worsens reperfusion injury. Methods: Single-cell RNA sequencing was performed on neutrophils isolated from the hearts of mice 24 hours post-MI/R. Additionally, mice underwent MI/R and heart tissues were harvested 6- and 24- hours after reperfusion for ELISA and qPCR analysis. Results: Utilizing single-cell RNA sequencing to better characterize the role of neutrophils and NETs in MI/R, we identified a significant population of neutrophils with a strong type I interferon (IFN) signature recruited to the area of infarct 24 hours post MI/R in a murine model. To further investigate the IFN response seen in this population of neutrophils, we preformed protein and gene expression analysis on heart tissue samples isolated from mice at timepoints in which neutrophils would be present in the area of infarct. Analysis by qPCR shows an increased fold change in gene expression of IFNα (7.22 ± 6.05, n = 3; 3.83 ± 2.22, n = 4) and IFNβ (6.74 ± 5.39, n = 3; 3.64 ± 2.25, n = 4) at 6- and 24- hours post MI/R respectively, compared to sham mice. Furthermore, analysis by ELISA shows a significant increase in IFNβ protein levels in the heart after 6- hours (0.41pg/mg ± 0.04, n =8) compared to sham mice (0.16 pg/mg ± 0.06, n = 7; p ≤ 0.01, Student’s t-test). Conclusions: These data show a rapid increase in type I IFN in the hearts of mice post-MI/R, which implicates neutrophils may be participating in the IFN signaling pathway. Identifying neutrophils as an early source of IFN can provide a potentially new therapeutic target in treating MI/R.

E3 ligase RNF5 inhibits type I interferon response in herpes simplex virus keratitis through the STING/IRF3 signaling pathway.

Herpes simplex keratitis (HSK), caused by the herpes simplex virus 1 (HSV-1), is a major blinding disease in developed countries. HSV-1 can remain latent in the host for life and cannot be eradicated. The infection causes the secretion of various cytokines and aggregation of inflammatory cells. In the early stage of inflammation, mainly neutrophils infiltrate the cornea, and CD4+ T cells mediate the immunopathological changes in herpetic stromal keratitis in the subsequent progression. The STING/IRF3-mediated type I interferon (IFN) response can effectively inhibit viral replication and control infection, but the activity of STING is affected by various ubiquitination modifications. In this study, we found that the expression of RNF5 was elevated in corneal tissues and corneal epithelial cells after infection with HSV-1. Immunofluorescence staining confirmed that RNF5 was mainly expressed in the corneal epithelial layer. We silenced and overexpressed RNF5 expression in corneal epithelial cells and then inoculated them with HSV-1. We found that the expressions of STING, p-IRF3, p-TBK1, and IFN-β mRNA increased after RNF5 silencing. The opposite results were obtained after RNF5 overexpression. We also used siRNA to silence RNF5 in the mouse cornea and then established the HSK model. Compared with the siRNA-control group, the siRNA-RNF5 group showed significantly improved corneal inflammation, reduced clinical scores and tear virus titers, and significantly increased corneal IFN-β expression. In addition, the expressions of the proinflammatory cytokines IL-6 and TNF-α in the corneal tissue were significantly decreased, indicating that RNF5 silencing could effectively promote IFN-I expression, inhibit virus replication, alleviate inflammation, and reduce corneal inflammatory damage. In summary, our results suggest that RNF5 limits the type I IFN antiviral response in HSV corneal epithelitis by inhibiting STING/IRF3 signaling.

Is IFN expression by NK cells a hallmark of severe COVID-19?

Natural Killer cells (NK) are crucial in host defense against viruses. There are many unanswered questions about the immune system in COVID-19, especially the mechanisms that contribute to the development of mild or severe forms of the disease. Although NK cells may have an essential role in the pathogenesis of COVID-19, the mechanisms involved in this process are not yet fully elucidated. Here, we demonstrate that CD3-CD56+ NK cells frequency in the volunteers who recovered from mild COVID-19 (Mild CoV) presented a significant increase compared to the healthy control (HC) and individuals recovering from severe COVID-19 (Severe CoV) groups. Furthermore, distinct IFN profiles in recovered COVID-19 patients with mild or severe clinical forms of the disease were observed in the total NK cells (CD3-CD56+). In the first group, NK cells express increased levels of IFN-α compared to the severe CoV, while higher production of IFN-γ in severe CoV was found. Moreover, NK cells in mild CoV express more cytolytic granules depicted by granzyme B and perforin. Compared to HC, PBMCs from mild CoV presented higher Ki-67 and TIM-3 production after Pool CoV-2 and Pool Spike CoV-2 peptides stimulus. In addition, non-stimulated PBMCs in the mild CoV group had higher NK TIM-3+ frequency than severe CoV. In the mild CoV group, Pool Spike CoV-2 and Pool CoV-2 peptides stimuli elicited higher granzyme B and perforin coexpression and IFN-α production by PBMCs. However, in severe CoV, Pool Spike CoV-2 reduced the coexpression of granzyme B, perforin, and CD107a suggesting a decrease in the cytotoxic activity of NK cells. Therefore, our study shows that NK cells may have a crucial role in COVID-19 with the involvement of IFN-α and cytotoxic properties that aid in developing qualified immune responses. Furthermore, the data suggest that higher amounts of IFN-γ may be linked to the severity of this disease.

021 CXCL13-producing peripheral T helper cells as potential mediators of photosensitivity in dermatomyositis

Dermatomyositis (DM) is a rare autoimmune disease that causes photosensitivity, disfiguring rash, and pruritus. Type I interferons (IFN-Is) are expressed in lesional (L) skin of DM, suggesting their role in disease pathogenesis. Using suction blistering on both L and non-lesional (NL) skin of treatment-naïve patients with DM followed by single-cell RNA sequencing (scRNA-seq) and flow cytometry, we found a distinct population of T cells expressing high levels of CXCL13, only in L DM. Their immunophenotype (CD4+/PD-1hi/CXCR5-) was characteristic of T peripheral helper (Tph) cells, a new subset of T cells recently described to drive autoimmunity in rheumatoid arthritis via CXCL13 mediated recruitment of B cells to synovium. However, consistent with previous reports, we did not find any B cells infiltrating L DM skin. Intriguingly, we observed high expression of CXCR5 on a subset of CD8+ T cells in both DM blood and L skin, suggesting CXCL13 promotes recruitment of cytotoxic CD8+ T cells to the inflamed skin. ELISA on the interstitial skin fluid confirmed higher levels of CXCL13 in L DM than NL and healthy (p<0.001), and immunohistochemistry highlighted CXCL13 staining colocalizing within perivascular CD4+ T cells. Using gene set enrichment analysis in our scRNA-seq data, we identified a robust IFN-I signature in Tph cells in DM skin. Further, we found that CD4+ T cells from DM blood displayed a significantly higher CXCL13 expression in response to IFNA than those from healthy subjects in vitro (p<0.01). Moreover, we observed that keratinocytes (KCs) isolated from L DM exhibit an enhanced IFNA expression in response to UVB than healthy in vitro, indicating an exaggerated IFN-I response to UVB in DM KCs induces differentiation of CXCL13-producing Tph cells in the skin, leading to the recruitment of circulating cytotoxic CD8+ T cells. These findings suggest a novel role for the IFN-I/CXCL13 axis in DM pathogenesis and its potential contribution to photosensitivity.

LB868 Th2 skewing promotes the expression of skin-homing molecules on T cells and is required for the induction of skin lesions in lupus-prone mice

Cutaneous lupus erythematosus (CLE) is an autoimmune skin disease characterized by high IFN expression. Recent advances have challenged the Th1-Th2 paradigm in CLE, as there are reports of expression of both Th1 and Th2 cytokines in lesional skin. We assessed the role of T cell skewing in the initiation of skin disease using a lupus-prone mouse model of CLE that depends on recognizing cognate antigen by infiltrating T cells in an immune environment perturbed by a TLR7-driven type I IFN response.

Avian IRF1 and IRF7 Play Overlapping and Distinct Roles in Regulating IFN-Dependent and -Independent Antiviral Responses to Duck Tembusu Virus Infection

Avian interferon regulatory factors 1 and 7 (IRF1 and IRF7) play important roles in the host’s innate immunity against viral infection. Our previous study revealed that duck tembusu virus (DTMUV) infection of chicken fibroblasts (DF1) and duck embryo fibroblasts (DEFs) induced the expression of a variety of IFN-stimulated genes (ISGs), including VIPERIN, IFIT5, CMPK2, IRF1, and IRF7. IRF1 was further shown to play a significant role in regulating the up-expression of VIPERIN, IFIT5, and CMPK2 and inhibiting DTMUV replication. In this study, we confirm, through overexpression and knockout approaches, that both IRF1 and IRF7 inhibit DTMUV replication, mainly via regulation of type I IFN expression, as well as the induction of IRF1, VIPERIN, IFIT5, CMPK2, and MX1. In addition, IRF1 directly promoted the expression of VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling were undermined. We also found that non-structural protein 2B (NS2B) of DTMUV was able to inhibit the induction of IFN-β mRNA triggered by Newcastle disease virus (NDV) infection or poly(I:C) treatment, revealing a strategy employed by DTMUV to evade host’s immunosurveillance. This study demonstrates that avian IRF7 and IRF1 play distinct roles in the regulation of type I IFN response during DTMUV infection.

Chicken TAX1BP1 suppresses type I interferon production via degrading chicken MAVS and facilitates infectious bursal diseases virus replication

Mammalian TAX1BP1 (TAX1 binding protein 1), originally identified as a partner of the HTLV-1 viral oncoprotein, functions in regulation of cellular cytokine production. TAX1BP1 plays an important signal transduction regulator, specifically modulating innate immune signaling pathways including NF–B and IRF3. The function of TAX1BP1, which regulates the innate immune response in mammals, has been well studied in previous reports, but the role of chicken TAX1BP1 (chTAX1) in IFN regulation and infectious bursal disease virus (IBDV) replication is still unclear. In this report, chTAX1 was successfully cloned and sub-inserted into a eukaryotic expression vector. The critical regions of chTAX1, such as LC3 binding motif, ubiquitin binding motif, are highly conserved compared to other organisms. We also found that chTAX1 inhibits IFN expression by promoting degradation of chicken MAVS (chMAVS). In addition, the distribution of chTAX1 altered and translocated to co-localize with both VP1 and VP3 after IBDV infection. Overexpression of chTAX1 promotes IBDV replication and knockdown of chTAX1 by RNA interference suppresses IBDV replication. In summary, our data initially indicate that chTAX1 is a suppressor of IFN expression as well as a promoter of IBDV replication.

Abstract 667: Inhibition of protein-protein interaction between STING and TRIM29 is a new approach to enhance anti-tumor immune response

Abstract Introduction: STimulator of INterferon Genes (STING) has been reported to be essential for antitumor immunity by inducing type I IFN expression, resulting in activation of both innate and adaptive immunity. Thus, pharmacological activation of STING pathway is actively being tested in many clinical studies. However, the clinical efficacy was not that clear, probably due to the limited expression of STING protein, which is tightly regulated by TRIM29 (E3 ligase)-mediated protein degradation pathway. Here, we present a novel small-molecule drug candidate with strong antitumor response in murine syngeneic model by upregulating cellular levels of STING through the inhibition of protein-protein interaction. Methods: The inhibitors of STING-TRIM29 interaction were identified using luciferase-complementation high-throughput screening system. Target protein of the inhibitor was identified in cellular thermostability shift assay. Their immunological activity was tested in Raw264.7 and A431 cells as well as CT26 murine syngeneic model. Results: The lead compound, SB24011, bound to STING and upregulated cellular levels of STING by inhibiting ubiquitin-mediated degradation. Together with STING agonist, SB24011 synergistically enhanced the inflammatory cytokine expression in cell lines as well as in CT26 syngeneic animal model. Intratumoral injection of SB24011 showed strong abscopal activity as single agent as well as synergistic antitumor responses with anti-PD1 antibody in CT26 syngeneic animal model. Conclusions: We identified novel small-molecule drug candidate, SB24011, which prevents the degradation of both murine and human STING proteins, resulting in enhanced immune responses together with STING agonist, cGAMP. SB24011 showed strong abscopal antitumor activity as single agent and synergistic antitumor activity as combination agent with other IO agents in CT26 syngeneic models. Citation Format: Seung Bum Park, Wansang Cho, Won Sol Chan, young il choi, Sungoh Ahn, Dong-Sup Lee. Inhibition of protein-protein interaction between STING and TRIM29 is a new approach to enhance anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 667.

Abstract 3276: Overcoming immune checkpoint blockade resistance via EZH1/2 dual inhibition by HM97662 in KRAS/LKB1 mutated NSCLC

Abstract Immune checkpoint blockades (ICBs) are known as a promising treatment option against in advanced non-small-cell lung cancer (NSCLC). And KRAS is the most frequently mutated oncogene in NSCLC. However, KRAS mutation is not a significantly associated with survival benefit of ICBs. Mutations in LKB1 (aka STK11) and frequently co-occurring KRAS mutations are accompanied with poor survival in metastatic NSCLC immuno-oncology trials. Even tumor mutational burden is a proposed-potential biomarker for response to ICBs, there are therapeutic unmet needs in NSCLC patient with KRAS/LKB1 (KL) mutation. Epigenetic regulation lead to enhanced anti-tumor immunity of innate immune responses via inducing type I interferons. Also it drives to enhance adaptive immune responses by targeting T cell recognition factors as well as recruitment immune cells to tumor microenvironments (TMEs). Thus, we demonstrated the pharmacological inhibition of Enhancer of zeste homolog (EZH)1/2 enhance the efficacy of clinically available immunotherapies in KRAS/LKB1 mutated NSCLC. Previously, we reported that a novel EZH1/2 dual inhibitor, HM97662, has showed potently decreased global H3K27me3 and strong anti-proliferative activities against various hematological cancer cell lines with EZH2 activating mutations as well as solid tumor cell lines with negatively mutated components of regulatory protein complexes. In this study, we evaluated that HM97662 could induce of STING expression and STING-dependent cytokine production in KL mutant NSCLC cell lines. Furthermore, we explored anticancer effect by inducing immune modulation by EZH2 inhibitors in combination with an anti-PD-(L)1 agent was evaluated. As a result, HM97662 potently restored STING expression in KL mutated cell lines such as A549 and H460, but did not KRAS/p53 (KP) mutated H358 cells. HM97662 also dose-dependently increased the mRNA expression of CXCL10, IFN-α and IFN-β and secretion of CXCL10, IFN-α, CCL2, CCL3 and CCL5 at nanomolar concentrations in H460 cells. In accordance with this, HM97662 showed potent growth inhibition against H460 cells under co-culture condition with activated T cells. Moreover, HM97662 had superior anti-proliferation efficacy among the tested compounds in H460 cells with activated T cells, when it combined with anti-PD-L1 antibody avelumab. In our exploratory study, HM97662 increments the sensitivity of immune checkpoint inhibitor by restoration of STING expression and STING pathway related chemo/cytokine production in KRAS/LKB1 mutated NSCLC cell lines. These results demonstrate that HM97662 drives anti-cancer effect by immune modulation in combination with anti-PD-(L)1 agent, suggesting therapeutic potency to combination with ICBs by turning non-inflamed to inflamed TME. Clinical trials to prove the effectiveness of HM97662 identified in preclinical studies need to be carried out immediately. Citation Format: Jooyun Byun, DongJin Hong, Miyoung Lee, Soonho Kweon, Seung Hyun Jung, Yu-Yon Kim, Hyunjin Park, Junghwa Park, Young Gil Ahn, Young Hoon Kim, Kwee Hyun Suh. Overcoming immune checkpoint blockade resistance via EZH1/2 dual inhibition by HM97662 in KRAS/LKB1 mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3276.

Abstract 2091: Macrophage PI3Kg control of chromatin accessibility and transcription factor activity promotes immune suppression and tumor progression

Abstract Tumor development and progression are associated with immune suppression and evasion as well as angiogenesis, fibrosis, and metastasis. Macrophages play critical roles in tumor progression by controlling these events and are the focus of efforts to develop novel cancer therapeutics. We previously showed that PI(3)Kinase γ, a class Ib phosphatidyl inositol 3, 4, 5 kinase that is expressed primarily in myeloid lineage cells, controls tumor progression by serving as a critical switch between immune stimulation and suppression. PI3Kγ also promotes angiogenesis, fibrosis and metastasis. PI3Kγ signaling in macrophages promotes transcription of tumor-promoting cytokines and factors such as Pdgf, Tgfb, Ccl2, Plau, Mmps and Arg-1 and inhibits expression of Il12, Tnfa, Nos2 and other pro-inflammatory factors to stimulate tumor progression and immune evasion. Genetic or pharmaceutical inhibition of PI3Kγ reverses these events and suppress tumor growth. In order to develop novel strategies to control macrophage contributions to cancer progression, we investigated in detail how this kinase controls gene expression. We found that PI3Kγ deletion or inhibition with a selective inhibitor IPI-549, which is currently in late stage clinical development for cancer therapy, stimulates the expression and secretion of IL12, IL6, IL1β, NOS, IFNα, and TNFα, while reducing the secretion and expression of IL10, CCL2, PDGFβ, ARG-1, TGFβ and other factors. Gene expression analysis indicates that PI3Kγ inhibition in macrophages rapidly stimulates transcription of NFκB and IFNγ driven pro-inflammatory pathways as well as nuclear translocation of transcription factors STAT1, IRF1, IRF3, TBK1 and NFκB and inhibition of CEBPβ nuclear activity. To further understand the impact of PI3Kγ on transcription in macrophages, we performed ATAC seq on WT and PI3Kγ null macrophages stimulated with LPS/IFNγ in order to determine the impact of PI3Kγ activity and inhibition on chromatin accessibility. We found that PI3Kγ deletion promoted more open chromatin sites for IRF, FRA I, Pu.1, and NFκB binding, as well as fewer open chromatin sites for AP1 (FOS/JUN), IRF8, and CEBP sites. Importantly, we identified overall chromatin accessibility in the promoters of some NFκB regulated genes, such as Tnfα and Il6, as well as novel open chromatin sites in the promoters for Il12b and Il1b in PI3Kγ null macrophages. We also identified novel closed chromatin sites in the promoters of Il10, Plau and Ccl2, genes that are downregulated upon PI3Kγ inhibition. Further studies to identify the mechanisms by which PI3Kγ regulates transcription are investigating PI3Kγ controls of epigenetic modifiers and transcription factors are ongoing. These studies should lead to the identification of new approaches to regulate tumor associated macrophages to inhibit tumor progression. Citation Format: Hui Chen, Jingwen Liao, Christina Flores, Diana Hargreaves, Judith Varner. Macrophage PI3Kg control of chromatin accessibility and transcription factor activity promotes immune suppression and tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2091.

Abstract 3297: IFNalpha by in vivo-engineered macrophages abates liver metastases and triggers counter regulatory responses limiting efficacy

Abstract The liver hosts an immune suppressive environment favouring metastatic seeding and proliferation of cancer cells. Pharmacological treatments, including immunotherapies, fail in the presence of liver metastases (LMS). Therefore, identifying new interventional tools and key targetable players involved in the immunosuppressive environment is of pivotal importance. We developed a lentiviral vector (LV)-based platform for selective genetic engineering of resident and tumour-associated macrophages enabling locally-sourced delivery of therapeutic molecules to LMS. Selective transgene expression is driven by a macrophage specific promoter and fine-tuned by microRNA target sequences. Upon systemic delivery of the LV, we observed enhanced transgene expression in macrophages located in areas surrounding LMS. We then equipped the LV with an IFNα-coding sequence, a cytokine with pleiotropic immune effects. Long term analysis in mice showed LV dose-dependent, sustained and well-tolerated IFNα expression. To investigate the therapeutic efficacy, we employed a colorectal cancer (CRC) organoid-based syngeneic mouse model of LMS containing molecular and histopathological hallmarks of the human disease. IFNα LV treatment significantly delayed LMS growth reaching a complete response in up to 50%. Single cell omics of LMS from IFNα LV-treated mice showed upregulation of IFNα-responsive genes, macrophage skewing to an antigen presenting (M1-like) polarization state, and expansion as well as reduced exhaustion of LMS-associated antigen specific CD8 T cells. Employing spatial transcriptomics, we found that the interface between LMS and liver parenchymal tissue was the major site of IFNα action, which was associated with enhanced immune activation and antigen presentation. Furthermore, we observed decreased angiogenesis and hypoxia in IFNα LV-treated LMS. When comparing LMS of treatment responsive to resistant mice, we found accumulation of activated CD8 T-cells in responsive whereas a high number of immunosuppressive T regulatory type 1 (TR1)-like cells in resistant mice. Molecular analyses suggest that TR1-like cell infiltration was associated with increased IL10 signaling in resistant mice. Furthermore, we found a positive correlation between IFNα and TR1-like cell signatures in human LMS and primary CRC thus supporting the link between IFNα activation and expansion of TR1-like cells in cancer. In summary, we developed an innovative gene-based platform that upon a single well-tolerated intravenous LV infusion rapidly promotes a protective therapeutic response against LMS through enabling immune activation. However, we also found that TR1-like cells might promote tumor immune evasion in presence of IFNα signaling in this setting, suggesting targeting of TR1-like cells when facing resistance to cancer immunotherapies that trigger IFNα signaling. Citation Format: Thomas Kerzel, Stefano Beretta, Eloise Scamardella, Chiara Balestrieri, Tamara Canu, Federica Pedica, Rossana Norata, Lucia Sergi Sergi, Marco Genua, Ostuni Renato, Anna Kajaste-Rudnitski, Antonio Esposito, Masanobu Oshima, Giovanni Tonon, Francesca Sanvito, Mario Leonardo Squadrito, Luigi Naldini. IFNalpha by in vivo-engineered macrophages abates liver metastases and triggers counter regulatory responses limiting efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3297.