IFN Signaling Research Articles

Interferon Regulatory Factor 7-mediated induction of interferon-α is required for survival from alphavirus encephalomyelitis

Abstract Alphaviruses are a group of arthropod-borne viruses that have the capacity to cause encephalomyelitis, which is inflammation of the brain and spinal cord due to the immune response to viral infection, and can result in fever, headache, and neurologic disease such as cognitive impairment, seizures, ataxia, and paralysis. Neurons are the primary target cells of Sindbis virus (SINV), the prototypic alphavirus; however, due to the indispensable nature of neurons, mechanisms of viral clearance from the central nervous system (CNS) during recovery cannot depend on immune-mediated killing of infected neurons and is therefore specialized. We have previously shown that interferon (IFN) regulatory factor (IRF) 7, a transcription factor important for amplification of type I IFN (mainly IFNα), is required for survival from infection with SINV. Mice deficient in IRF7 succumb to infection – despite local production of IFNβ – while wild type (WT) mice recover. Treatment of Irf7−/−mice with IFNα rescues the WT phenotype, suggesting that IRF7-mediated induction of IFNα and subsequent downstream signaling is required for survival from SINV infection. Additionally, Ifnb−/−mice do not phenocopy Irf7−/−mice, further suggesting that IFNα is required for survival while IFNβ is dispensable despite usage of the same receptor. Interestingly, Ifnb−/−mice have similar viral titers to Irf7−/−mice, implying that the protective effects of IFNα are independent from restriction of viral replication. These findings show subtype-specific differences in protection by type I IFN and can inform treatment options for alphavirus-induced encephalomyelitis. These works have been funded by the U.S. National Institutes of Health (R01 NS087539 and T32 AI007417).

Type I and II interferons induce granulopoiesis and correlate with organ damage during infection-induced shock

Abstract The mechanisms that drive pathology in infection-induced shock are not well understood. The tick-borne pathogen Ixodes ovatuserhlichia (IOE) induces severe inflammation, vascular damage, liver injury, and death. Mice that lack the receptor for type I IFN, or receptors for both type I and II IFNs, improves survival by 50–70 and 90%, respectively. IOE infection induced a significant increase in circulating granulocytes that depended on both type I and II IFNs. We also observed an IFN-dependent increase in eosinophils and basophils in the bone marrow in IOE-infected C57BL/6TA (WT). Although IOE infection induced an increase in bone marrow IL-5 and GM-CSF levels independent of IFN signaling, bone marrow concentrations of IL-3 and IL-13, both known to promote eosinophil differentiation and survival, increased in an IFNAR-dependent manner. Furthermore, we demonstrate an infection- and IFN-dependent increase of CCL3 and CCL5, two chemokines that promote eosinophil activation and migration. While bone marrow and blood eosinophils and basophils were increased, we observed a specific depletion of eosinophils in WT liver at day 7 post-IOE infection, correlating with increased cell death and severe liver pathology. In contrast, basophil frequencies were increased in IOE-infected WT mice. Additionally, Ifnar-deficient and Ifnar-Ifngr-deficient mice exhibited normal eosinophil and basophil frequencies and reduced liver inflammation, similar to uninfected mice. Together, our data reveal an IFN-driven cytokine and chemokine response that correlates with rampant granulopoiesis and mortality and provide evidence that IFN-induced eosinophil activation may contribute to vascular leak and liver damage in infection-induced shock. Supported by grants from NIH R35GM131842

Targeted activation of IL-22 producing innate lymphoid cells enhances host resistance against Clostridioides difficileinfection

Abstract Clostridioides difficile, the most common hospital acquired pathogen in the United States, infects the gastrointestinal tract following perturbation of the intestinal microbiota causing debilitating, potentially fatal colitis. Primary C. difficile infection has a high recurrence rate following antibiotic treatment, emphasizing the need to develop alternative treatment strategies to reduce the burden on the healthcare system. Innate lymphoid cells (ILCs) are necessary for acute host defense following C. difficile infection and represent a promising therapeutic target to harness the patient’s innate immune defenses to limit disease. Here, using a murine model of C. difficile infection, we report that oral administration of Resiquimod (R848), a TLR-7 agonist previously reported to activate type-3 ILCs (ILC3s), protects mice from lethal C. difficile challenge. R848 treatment did not alter intestinal microbial communities or impact C. difficile burden. R848 treatment conveyed protection via activation of the host immune system in a TLR-7 dependent manner. R848-mediated protection was independent of T and B cells as well as type-I IFN signaling. Mice lacking ILCs, however, did not respond to R848 treatment and remained susceptible to infection, identifying these cells as the key targets of R848 activity. Last, R848 treatment robustly induced ILC3s in the large intestine to produce IL-22 and genetic ablation of IL-22 abrogated R848-mediated protection from C. difficile infection. Combined, these data identify an immunostimulatory molecule that activates the IL-22 producing ILC3s to support host defense against C. difficile infection without causing further dysbiosis of the intestinal microbiota. NIH R01AI158830, R21AI164385

USP18 promotes endometrial receptivity via the JAK/STAT1 and the ISGylation pathway.

Interferon-tau (IFNT), a pregnancy recognition signal in ruminants, promotes the establishment of endometrial receptivity by inducing the expression of interferon-stimulated genes (ISGs) via the Janus kinase/signal transducer and activator of transcription (JAK/STATs) signaling pathway. However, the precise mechanisms remain largely unknown. Ubiquitin-specific protease 18 (USP18) acts specifically on the ISGylation modification system to exert deubiquitination and participates in the regulation of the type I IFN signaling pathway. The purpose of this study was to determine the role and mechanism of USP18 on endometrial receptivity in goat. USP18 was mainly localized in the uterine luminal and glandular epithelium, and its expression levels were significantly increased from days 5-18 of early pregnancy. Progesterone (P4), estradiol (E2), and IFNT significantly stimulated USP18 expression in goat endometrial epithelial cells (gEECs) cultured invitro. Meanwhile, the markers of endometrial receptivity HOXA11, ITGB1, ITGB3, and ITGB5 were significantly upregulated after USP18 overexpression in gEECs. However, USP18 interference significantly inhibited the expression of HOXA10, ITGB1, ITGB3, and ITGB5 in gEECs. In addition, both the phosphorylation levels of STAT1 and the expression of ISGylation-modified proteins were significantly increased after USP18 silencing in gEECs. Furthermore, pretreatment with the STAT1 inhibitor Fludara markedly restored the effect of USP18 interference in gEECs. In summary, USP18 may play an important role in promoting goat endometrial receptivity by regulating the JAK/STAT1 pathway and ISGylation.

Abstract 514: Inhibiting The P2Y12 Receptor In Megakaryocytes And Platelets Suppresses Interferon-associated Inflammatory Responses

Megakaryocytes (MKs) and platelets are recognized as mediators of inflammation and immunity. Antiplatelet therapies are widely used to prevent cardiovascular events; however, their role in attenuating platelet-mediated inflammation is uncertain. We investigated whether antiplatelet drugs, aspirin (ASA) and P2Y 12 inhibitors, alter the MK and platelet transcriptomes. We hypothesized that antiplatelet therapy reduces MK- and platelet-mediated inflammation. RNA-seq was performed in CD34 + derived MKs treated with ASA (100 μM), P2Y 12 inhibitor (AZD1283, 5 μM) or PBS for 24 hours. We validated our MK model using RNA-seq data from platelets of healthy volunteers randomized in a crossover design to low-dose ASA (n = 70, 64% female, 43.6 ± 9 years) or P2Y 12 inhibitor (ticagrelor, n = 57, 61% female, 43.9 ± 10 years) for 4 weeks each. The clinical relevance of our findings was assessed using RNA-seq data from platelets of SLE patients (n = 54, 100% female, 40.6 ± 12 years) and matched controls (n = 18, 100% female, 42.1 ± 15 years). Finally, we validated our findings in vitro using RT-qPCR and western blot. We identified 74 ASA- and 2,499 P2Y 12 inhibitor MK-responsive genes. In contrast to ASA, P2Y 12 inhibitor-mediated gene changes between MK and platelet transcriptomes were positively correlated (R=0.37, p<0.001). P2Y 12 inhibition downregulated IFNα response pathway in both MKs (NES = -2.3, p = 0.002) and platelets (NES = -2.24, p = 3.22 х 10 -8 ). SLE is characterized by activation of the IFN system. Consistently, the IFNα pathway was upregulated in platelets from SLE patients versus controls (NES = 3.38, p = 1.25 х 10 -9 ). Among 72 upregulated IFNα genes in SLE patients, 33 were inhibited with P2Y 12 inhibition in MKs or platelets, including IRF7 and IFITM3. Finally, we validated the P2Y 12 inhibitory effect on IFN-associated responses. While IFNα (1000 U/mL) significantly upregulated both IRF7 and IFITM3, pre-treatment with P2Y 12 receptor inhibition attenuated this response on both, the transcriptomic and proteomic levels. Our data indicate that targeting the P2Y 12 receptor attenuates MK- and platelet-mediated IFN signaling pathways. P2Y 12 -mediated suppression of platelet-induced IFNα signaling may be beneficial in inflammatory and autoimmune diseases including SLE.

3D co-culture models demonstrate radiation-mediated activation of tumor-associated macrophages

Abstract Type I IFN signaling mediated by STING pathway is important for the induction of radiation-stimulated adaptive immune responses in responsive tumors. Macrophages recruited to tumors following radiotherapy (RT) can influence the response to RT. Reprogramming immunosuppressive M2-like macrophages in tumors to exhibit a more activated M1-like phenotype can help improve response to therapies. Here, we hypothesize that a 3D co-culture model of cancer cells and macrophages will be more contextually relevant to the events occurring in tumors and can be used to study the crosstalk that occurs between these cells in vivo. We initially optimized seeding densities of murine MC38 colorectal carcinoma cells to develop spheroids of consistent size. Using GFP+ macrophages and Hoechst stained MC38, we imaged the development and localization of these cells during spheroid formation. Next, co-culture spheroids were exposed to 12 Gy radiation on day 1. Viability and phenotype of macrophages in spheroid co-cultures was assessed on day 4 using flow cytometry by staining for CD80, CD86, MHC Class II, CD69 and CD206 expression. STING ligand, cyclic di-AMP (CDA) treated spheroid co-cultures were used as positive controls. CDA treatment significantly increased CD80/86 expression in 3D co-cultures. Treatment with 12 Gy radiation showed similar effect. Interestingly, macrophages derived from MyD88 Lyz2Cre, MyD88 fl/fland STING knockout mice also showed a significant increase in CD80/86 and CD40, suggesting that macrophage activation was independent of MyD88 or STING signaling. Ongoing studies aim to explore the pathways activated on irradiation in these cells and further cross-validate with the macrophage phenotypes observed in vivo in response to RT.

Analysis of intestinal epithelial cell responses to IFN-γ during Cryptosporidiuminfection

Abstract Cryptosporidium species are intracellular parasites that infect intestinal epithelial cells (IEC) and the rapid cycle of growth, cell lysis and reinfection every 12 hours can cause severe enteric disease in young or immunocompromised patients. Interferon-γ (IFN-γ) has a crucial role in protective immunity and mice with an IEC-specific deletion of the IFN-γ receptor (IFN-γR ΔIEC) are highly susceptible to infection. However, the dynamics of IFN-γ signalling to IEC and how this leads to parasite control remain poorly understood. Based on the use of a reporter mouse for IFN signalling in vivo, while macrophages showed a transient response to treatment with IFN-γ, IEC exhibited a sustained response to IFN-γ that peaks at 24h. Indeed, the treatment of mice with recombinant IFN-γ transiently limits parasite shedding but takes 24h to show protective activity. Single-cell RNA sequencing of enterocytes from naïve and infected mice identified upregulation of several IFN-γ-inducible effectors but highlighted that induction of these genes was comparable in infected and bystander IEC. Together, these results suggest that while Cryptosporidium-infected cells are responsive to IFN-γ signalling, the protective effects of IFN-γ may be mediated through activation of uninfected bystander enterocytes. These studies provide new insights into the dynamics and function of IFN-γ signalling on IEC that are broadly relevant to other IEC-restricted pathogens. Supported by grants from NIH (R01 AI148249) and post-doctoral fellowships from the Canadian Institutes for Health Research and Fonds de Recherche du Québec – Santé.

Secondary cues alter ISG protein repertoires to unlock antiviral subprograms triggered by interferons

Abstract Immune defenses are triggered by cytokines like interferon-α (IFN-α) and IFN-γ in diverse cells. Both IFNs induce a class of factors termed IFN-stimulated genes (ISGs). Many ISG proteins have antiviral functions. There is a growing appreciation that ISG repertoires are cell-type specific and that this bias impacts infection outcomes. However, the cues and mechanisms driving these variable responses are ill-defined. Given key roles for immunometabolism in immune cell activation, we hypothesized that local cues like metabolites shape tissue specific ISG defenses. To model the impact of metabolic cues on host defenses in non-immune cells, we infected human cells with two unrelated viruses: vaccinia virus, the prototypical poxvirus, and herpes simplex virus-1 (HSV-1). Unexpectedly, we find that cells favoring glycolysis, but not OXPHOS, catalyze potent IFN-γ restriction of vaccinia and HSV-1 that attenuate viral-gene and protein expression as well as infectious virus production. This response is specific as modulating IFN-α signaling by supplementation with either glycolysis or OXPHOS cues has no effect on viral replication. This interferon-induced subprogram is independent of differences in canonical IFN-γ signal transduction, occurs at low levels of IFN-γ, and induced by physiological levels of glucose. RNA-seq reveals no marked changes in the “usual suspects” (e.g. ISGs). Instead, we find secondary cues regulate ISG protein levels. Deletion of a single ISG protein, which is differentially stabilized by secondary cues, is sufficient to rescue vaccinia replication. These data indicate that secondary cues can alter infection outcomes in the same cell-type by unlocking subprograms embedded in interferon catalyzed responses.

Triple immune modulator therapy for aberrant hyperinflammatory responses in severe COVID-19

A dysregulated hyperinflammatory response is a key pathogenesis of severe COVID-19, but optimal immune modulator treatment has not been established. To evaluate the clinical effectiveness of double (glucocorticoids and tocilizumab) and triple (plus baricitinib) immune modulator therapy for severe COVID-19, a retrospective cohort study was conducted. For the immunologic investigation, a single-cell RNA sequencing analysis was performed in serially collected PBMCs and neutrophil specimens. Triple immune modulator therapy was a significant factor in a multivariable analysis for 30-day recovery. In the scRNA-seq analysis, type I and II IFN response-related pathways were suppressed by GC, and the IL-6-associated signature was additionally downregulated by TOC. Adding BAR to GC and TOC distinctly downregulated the ISGF3 cluster. Adding BAR also regulated the pathologically activated monocyte and neutrophil subpopulation induced by aberrant IFN signals. Triple immune modulator therapy in severe COVID-19 improved 30-day recovery through additional regulation of the aberrant hyperinflammatory immune response.

Generation of an A549 ISRE-luciferase stable cell line

With its human lung origin, A549 cell line is a designated cellular model for viral respiratory infections studies. As such infections are known to lead to innate immune responses, various IFN signaling modifications occur in infected cells and have to be considered in respiratory viruses experiments. Here, we describe the generation of an A549 stable cell line that expresses firefly luciferase upon interferon-β stimulation, as well as upon RIG-I transfection and upon influenza A virus infection. Of the 18 clones generated, the first one, namely A549-RING1, demonstrated appropriate luciferase expression in the different conditions tested. This newly established cell line may therefore be used to decipher the impact of viral respiratory infection on innate immune response depending on IFN stimulation, without any plasmid transfection step. A549-RING1 can be provided upon request.

African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

This study evaluates the role of the late viral protein, pA104R, in African swine fever virus immunosuppression. ASFV-encoded pA104R is a putative histone-like protein that is highly conserved throughout different virulent and non-virulent isolates. Previous studies have demonstrated that pA104R plays a vital role in the ASFV replication cycle and is a potential target for antiviral therapy. Here, we demonstrated that pA104R is a potent antagonist of type I interferon signaling. IFN-stimulated response element activity and subsequent transcription of co-transfected and endogenous interferon-stimulated genes were attenuated by pA104R treatment in HEK-293 T cells. Immunoprecipitation assay and reciprocal pull-down showed that pA104R does not interact directly with STAT1, STAT2, or IRF9. However, pA104R could inhibit IFN signaling by attenuating STAT1 phosphorylation, and we identified the critical amino acid residues (R/H69,72 and K/R92,94,97) involved through the targeted mutation functional assays. Although pA104R is a histone-like protein localized to the nucleus, it did not inhibit IFN signaling through its DNA-binding capacity. In addition, activation of the ISRE promoter by IRF9-Stat2(TA), a STAT1-independent pathway, was inhibited by pA104R. Further results revealed that both the transcriptional activation and recruitment of transcriptional stimulators by interferon-stimulated gene factor 3 were not impaired. Although we failed to determine a mechanism for pA104R-mediated IFN signaling inhibition other than attenuating the phosphorylation of STAT1, these results might imply a possible involvement of epigenetic modification by ASFV pA104R. Taken together, these findings support that pA104R is an antagonist of type I interferon signaling, which may interfere with multiple signaling pathways.

Abstract 4430: Sacituzumab-Interferon beta mutein fusion protein, ABN202 (anti Trop2-interferon beta mutein), is a potent therapeutics for Trop-2-positive urothelial cancer

Abstract Background: Trop2, trophoblast cell-surface antigen 2, is a type 1 transmembrane glycoprotein overexpressed in many solid tumors such as breast, lung, pancreatic, gastric, colon, urothelial cancer. Overexpression of Trop2 is associated with aggressive disease progression and poor prognosis. Recently, Trop2 targeting antibody-drug conjugate such as sacituzumab-govitecan (Trodelvy®) was approved for the treatment of triple-negative breast cancer and urothelial cancer (UC) patients. Interferon-beta (IFN-beta), a pleiotropic cytokine, can induce direct/in-direct tumor killing and activate anti-tumor immune response in tumor microenvironments. However, it is challenging to develop IFN-beta as an anti-cancer agent due to its low biophysical properties, short half-life, and the systemic toxicity. Previously, we developed IFN-beta mutein (ABN102), which showed improved stability and biophysical properties compared to wild-type IFN-beta. We hypothesis to reduce any toxicity of ABN102 by fusing with tumor targeting antibody. In this study, we generated antibody cytokine fusion protein (ABN202), anti Trop2-interferon-beta mutein, and evaluated the efficacy of ABN202 against Trop2-positive UC cell line models. Method & Result: We aimed to determine the direct anti-cancer efficacy of ABN202 using Trop2-positive UC cell lines and the indirect immune cell mediated effect of ABN202 using Trop2-positive UC cell lines co-cultured with human peripheral blood mononuclear cell (PBMC). The in vivo efficacy of ABN202 was further confirmed in Trop2 positive tumor mouse model. Our results revealed that ABN202 directly inhibited proliferation and activates the apoptosis-related signaling mechanisms via Type 1 IFN signaling, showing better efficacy than either anti Trop2 antibody (sacituzumab) or ABN102 alone. ABN202 induces the activation of PBMC subsets and enhanced immune cell-mediated cancer cell killing when UC cell lines were co-cultured with PBMC. The anti-cancer efficacy of ABN202 were confirmed in Trop2-positive tumor mouse model. Conclusion: We evaluated the direct and indirect anti-cancer efficacy of ABN202 (anti Trop2-interferon-beta mutein) in Trop2-positive urothelial cancer. Our results support that ABN202 is a potent drug candidate in Trop2-positive urothelial cancer by directly inhibiting tumor growth and activating anti-cancer immune responses through type 1 IFN signaling. Citation Format: Jiyang Lee, Myeung-Ryun Seo, Heegeon Park, YeongMun Kim, Saehyung Lee, Na Young Kim, Chan Gyu Lee, Hae Min Jeong, Jun Young Choi, Young kee Shin. Sacituzumab-Interferon beta mutein fusion protein, ABN202 (anti Trop2-interferon beta mutein), is a potent therapeutics for Trop-2-positive urothelial cancer. [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 4430.

Abstract 6410: Deciphering the role of MyD88 signaling pathway in regulating type I IFN-mediated responses to radiation therapy in solid tumors

Abstract The balance of innate signaling through adaptor proteins such as MyD88 and TRIF is critical in directing the pattern of inflammatory responses following exposure to endogenous adjuvants. While innate stimuli can cause inflammation that supports adaptive immunity, cancer myeloid cells can be pre-polarized such that the same inflammatory signals cause myeloid cells to suppress adaptive immune responses. We aim to investigate the signals regulating type I IFN secretion by innate immune cells in tumors in order to improve type I IFN secretion, activation of innate immune cells, direct macrophage polarization and improve CD8+ T cell mediated anti-tumor immunity. To better understand the role of MyD88-mediated signaling in driving response to innate adjuvants released following RT in solid tumors, we developed MyD88 conditional knockout mouse models. MyD88 was deleted specifically in CD11c expressing dendritic cells (DC), Lck expressing T cells, or Lyz2 expressing myeloid cells that include macrophages, monocytes and granulocytes. Mice bearing pancreatic tumors (Panc02-SIY or PK5L1940) were randomized to no treatment or 16 Gy CT-guided RT and followed for treatment responses. Lyz2-Cre/MyD88fl/fl mice demonstrated improved responses to RT in pancreatic tumors as compared to control MyD88fl/fl mice, or Lck-Cre mice. The improved responses in Lyz2-Cre/MyD88fl/fl mice were shown to be dependent on CD8+ T cells as well as on type I IFN signaling. To analyze mechanisms of response, tumors were digested into a single cell suspension and CD45+ cells were isolated for single cell sequencing three days post-RT. Lyz2-Cre/MyD88fl/fl mice showed diminished Th1 and Th2-type T cells and had a higher M1/TAM ratio compared to MyD88fl/fl mice. Loss of MyD88 in myeloid cells resulted in increased activation of IFN-dependent transcriptional responses in multiple immune cell populations in the tumor. To model responses ex vivo, bone marrow-derived macrophages (BMDMs) were activated with lipopolysaccharide (LPS) or a synthetic cyclic dinucleotide (CDA) for analysis of cytokine secretion in either monolayer or 3D spheroid culture conditions. BMDM derived from Lyz2-Cre/MyD88fl/fl cultured as a monolayer showed significantly altered secretion of TNF-α, IL-10 and IL-6 on activation with LPS as compared to controls. Preliminary data obtained from 3D spheroid culture systems demonstrated that 3D interactions between cancer cells and macrophages resulted in a significant increase in BMDM co-stimulatory phenotype following activation with innate stimuli compared to 2D cultures. These data suggest that conventional innate signaling through MyD88 in myeloid populations suppresses IFN production and adaptive immune control of irradiated tumors. 3D cultures are an effective tool to study cellular interactions ex vivo and screen novel interventions prior to in vivo translation. Citation Format: Aanchal Preet Kaur, Terry R. Medler, Tiffany C. Blair, Alejandro F. Alice, Megan R. Ball, Alexa K. Dowdell, Brian D. Piening, Marka R. Crittenden, Michael J. Gough. Deciphering the role of MyD88 signaling pathway in regulating type I IFN-mediated responses to radiation therapy in solid tumors [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 6410.

BET Inhibition Sensitizes Immunologically Cold Rb-Deficient Prostate Cancer to Immune Checkpoint Blockade.

Non-T-cell-inflamed immunologically "cold" tumor microenvironments (TME) are associated with poor responsiveness to immune checkpoint blockade (ICB) and can be sculpted by tumor cell genomics. Here, we evaluated how retinoblastoma (Rb) tumor-suppressor loss-of-function (LOF), one of the most frequent alterations in human cancer and associated with lineage plasticity, poor prognosis, and therapeutic outcomes, alters the TME, and whether therapeutic strategies targeting the molecular consequences of Rb loss enhance ICB efficacy. We performed bioinformatics analysis to elucidate the impact of endogenous Rb LOF on the immune TME in human primary and metastatic tumors. Next, we used isogenic murine models of Rb-deficient prostate cancer for in vitro and in vivo mechanistic studies to examine how Rb loss and bromodomain and extraterminal (BET) domain inhibition (BETi) reprograms the immune landscape, and evaluated in vivo therapeutic efficacy of BETi, singly and in combination with ICB and androgen deprivation therapy. Rb loss was enriched in non-T-cell-inflamed tumors, and Rb-deficient murine tumors demonstrated decreased immune infiltration in vivo. The BETi JQ1 increased immune infiltration into the TME through enhanced tumor cell STING/NF-κB activation and type I IFN signaling within tumor cells, resulting in differential macrophage and T-cell-mediated tumor growth inhibition and sensitization of Rb-deficient prostate cancer to ICB. BETi can reprogram the immunologically cold Rb-deficient TME via STING/NF-κB/IFN signaling to sensitize Rb-deficient prostate cancer to ICB. These data provide the mechanistic rationale to test combinations of BETi and ICB in clinical trials of Rb-deficient prostate cancer.

Abstract 6348: ABN202 (αEGFR-interferon-β mutein), a potent antibody-cytokine fusion protein for the treatment of EGFR-positive non-small cell lung cancer

Abstract ABN202 is an antibody-cytokine fusion protein consisting of interferon-β mutein (ABN102) molecularly fused to a anti-EGFR monoclonal antibody that recognizes the EGFR expressed on various types of cancer cells. In previous studies, we developed ABN102, a bio-better version of recombinant human interferon-β with hyperglycosylation via site-directed mutagenesis to improve stability, productivity, and pharmacokinetics properties. An addition of glycosylation at the N25 residue of ABN102 is expected to reduce the peripheral toxicity of ABN202, as it can shield the IFNAR2 binding interface. In this study, the binding ability and biological activity of ABN202 was evaluated in vitro models. ABN202 downregulates the EGFR protein expression and reduces EGFR phosphorylation by degrading target proteins via internalization in the EGFR-positive NSCLC cell line. In addition, we also determined the direct and indirect anti-cancer activity of ABN202 in preclinical models. Taken together, we suggest that ABN202 has potent anti-cancer activities, via direct cytotoxicity in EGFR-positive tumors and indirectly activating immune cells through Type I IFN signaling. we conclude ABN202 is a promising drug candidate for patients with EGFR-positive NSCLC. Citation Format: Heegeon Park, Saehyung Lee, Hayeon Park, Sunghyun Hong, Jaemun Kim, Sungyoul Hong, Ji-hyun Park, Young Kee Shin, Jun Young Choi, Na Young Kim. ABN202 (αEGFR-interferon-β mutein), a potent antibody-cytokine fusion protein for the treatment of EGFR-positive non-small cell lung cancer [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 6348.

Essential role of an ERV-derived Env38 protein in adaptive humoral immunity against an exogenous SVCV infection in a zebrafish model.

Endogenous retroviruses (ERVs) are the relics of ancient retroviruses occupying a substantial fraction of vertebrate genomes. However, knowledge about the functional association of ERVs with cellular activities remains limited. Recently, we have identified approximately 3,315 ERVs from zebrafish at genome-wide level, among which 421 ERVs were actively expressed in response to the infection of Spring viraemia of carp virus (SVCV). These findings demonstrated the previously unrecognized activity of ERVs in zebrafish immunity, thereby making zebrafish an attractive model organism for deciphering the interplay among ERVs, exogenous invading viruses, and host immunity. In the present study, we investigated the functional role of an envelope protein (Env38) derived from an ERV-E5.1.38-DanRer element in zebrafish adaptive immunity against SVCV in view of its strong responsiveness to SVCV infection. This Env38 is a glycosylated membrane protein mainly distributed on MHC-II+ antigen-presenting cells (APCs). By performing blockade and knockdown/knockout assays, we found that the deficiency of Env38 markedly impaired the activation of SVCV-induced CD4+ T cells and thereby led to the inhibition of IgM+/IgZ+ B cell proliferation, IgM/IgZ Ab production, and zebrafish defense against SVCV challenge. Mechanistically, Env38 activates CD4+ T cells by promoting the formation of pMHC-TCR-CD4 complex via cross-linking MHC-II and CD4 molecules between APCs and CD4+ T cells, wherein the surface subunit (SU) of Env38 associates with the second immunoglobin domain of CD4 (CD4-D2) and the first α1 domain of MHC-IIα (MHC-IIα1). Notably, the expression and functionality of Env38 was strongly induced by zebrafish IFNφ1, indicating that env38 acts as an IFN-stimulating gene (ISG) regulated by IFN signaling. To the best of our knowledge, this study is the first to identify the involvement of an Env protein in host immune defense against an exogenous invading virus by promoting the initial activation of adaptive humoral immunity. It improved the current understanding of the cooperation between ERVs and host adaptive immunity.

Abstract 6653: Analysis of selective response of anti-PD-L1 treatment in huPBMC-NCG mice reconstituted with different donors

Abstract Despite the success of programmed death ligand 1 (PD-L1) and programmed death 1 (PD-1) blockage as cancer therapies, the overall clinical response rates are still unsatisfying, with only a 25% response rate in most cancers. The underlying mechanisms of non-responsiveness towards anti-PD-L1 treatment have been extensively studied and associated with many factors, including PD1/PD-L1 expression level, tumor mutation burden, IFN signaling, loss of MHC-I, tumor microenvironment (immune infiltration, suppressive immune system), and so on. Immunodeficient mice such as the NSG and NCG are commonly used to study the efficacy of cancer immunotherapies. These mice, reconstituted with either human PBMC or HSC, act as an avatar to provide a window of the potential agent efficacy at the preclinical level. However, due to the challenge of obtaining an HSC/PBMC donor, and often cost and time can also be a limiting factor, many studies only use 2-5 human donors. Here we present data demonstrating the significant variation observed between donor-to-donor, highlighting the importance of testing multiple donors. We reconstituted immune system and tumor tissue humanized mouse models using peripheral blood mononuclear cells (PBMCs), and cell line-derived xenografts (CDX) in severe immunodeficiency NCG mice. Anti-tumor efficacy of anti-PD-L1 (Tecentriq) in huPBMC-NCG mice engrafted with several tumor cells including MDA-MB-231, HCC827, NUGC4, and some other CDX cell lines was tested in our system. In the cases of MDA-MB-231, HCC827, and NUGC4, different donors were enrolled in the same study, and tumor growth inhibition measured by tumor volume (TGITV) of 3% to 52.40% (MDA-MB-231), 5% to 33% (HCC827) and 4.75% to 20.74% (NUGC4) were observed. Especially, In the case of MDA-MB-231, 6 donors were enrolled in this study at the same time. About 40% of immune infiltration was first observed in tumors. High expression of PD1 on immune cells and PDL1 on tumor cells were also detected from different donors simultaneously. Although the similar immune infiltration and PD1/PDL1 expression, the TGITV of 6 donors varied between 3% and 52.40%, suggesting the donor-dependent response to anti-PD-L1 treatment, which is similar to the clinical trials. Our data showed diverse in vivo responses when multiple human PBMC donors were used for preclinical agent evaluation. While unsurprising, it showed the critical need to include various human PBMC donors when an agent is in preclinical status because the donors' immune profile may dictate the study's outcome. This can be divided into two parts, on the one hand, donor screening for favorable efficacy response donors in advance is an alternative approach to ensure the positive efficacy of anti-PDL1 blockade relative therapy. On the other hand, the low-efficacy response donors also can be candidates for anti-PD1/PDL1 combination therapy. Citation Format: Tingting Gu, Weiwei Yu, Hongyan Sun, Fang Zhu, Huiyi Wang, Mengting Wang, Shuai Li, Jianming Xu, Santi Chen, Zhiying Li, Mark Wade Moore, Cunxiang Ju, Hongyu Wang, Jing Zhao, Xiang Gao. Analysis of selective response of anti-PD-L1 treatment in huPBMC-NCG mice reconstituted with different donors [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 6653.

Abstract 1260: Tumor hypoxia conditions glioblastoma cells for immunosuppression

Abstract Glioblastoma (GBM) is the most common and lethal malignant brain tumor that invariably recurs after standard therapy, with a median survival of only ~16 months. A major determinant of high GBM lethality is the overall immunosuppressive tumor microenvironment and the presence of treatment resistant hypoxic niches. In a recent study, we engineered murine GBM cell line GL261 with a lentiviral HRE driven fluorescent reporter, HRE-UnaG-PEST to study the relationship between tumor hypoxia and immunosuppression. By 3-4 weeks post-transplant in mice, we observed an abundance of UnaG+ GBM cells that were organized as pseudopalisading patterns around necrotic cores, similar to histological features observed in GBM patient specimens. We also observed an accumulation of TAMs and cytotoxic T cells inside hypoxic cores surrounded by UnaG+ GBM cells, which coincided with a gradual increase in vascular disarray and development of hypoxic niches. Single cell RNA sequencing (scRNA-seq) revealed that UnaG+ GBM cells were negatively enriched for neuroinflammation, Type II IFN signaling, TNFα and NF-κB signaling pathways, and IFNα and IFNγ responses. Furthermore, the gene ontology term “Negative Regulation of Immune System Process” (including genes such as Mif, Lgals3, Sdc4, Cd44, Dusp1) was predominantly upregulated in UnaG+ GBM cells. Hypoxia transcriptional program in GL261 GBM thus favors immunosuppression. To longitudinally study the immunosuppressive transcriptional programs of hypoxic GBM cells, we combined lentiviral HRE-CreERT2 with a knock-in of CAG-loxP-STOP-loxP-tdTomato (LSL-tdTom) reporter into the Rosa26 locus as a lineage tracing reporter for hypoxia-exposed GBM cells. A subclone of GL261-HRE-CreERT2; LSL-tdTom cells that displayed low baseline expression and faithful induction of tdTomato upon exposure to 1% O2 and 4-hydroxytamoxifen was identified and intracranially transplanted in mice. After a single tamoxifen injection day 21 post-transplant, mice were traced for 2 days or 7 days before analysis. At both timepoints, we observed abundant tdTom+ GBM cells spread throughout the tumor. Few tdTom+ GBM cells showed GLUT1 expression, more so in 7-day tracing than 2-day tracing. Hence, as the tumor progressed, most tdTom+ GL261 cells no longer experienced hypoxia, likely due to new angiogenesis or migration away from hypoxic areas. No tdTom+ GL261 cells were observed in parallel control experiments where tamoxifen was not injected. To investigate if hypoxia-induced immunosuppressive adaptations are preserved along tumor progression, further scRNA-seq and FACS studies will be performed to characterize the transcriptional profile of tdTom+ GBM cells. To test if hypoxic exposure is a critical conditioning event that promotes an immunosuppressive environment in GBM, we plan to genetically eliminate hypoxic GBM cells by inducing expression of Diptheria Toxin and evaluate effect on functional state of immune cells. Citation Format: Anirudh Sattiraju, Sangjo Kang, Hongyan Zou, Roland Friedel. Tumor hypoxia conditions glioblastoma cells for immunosuppression [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 1260.

Abstract 1817: WTX-613, (JZP898) a selectively activated IFNα INDUKINE™ molecule, reprograms the tumor microenvironment and generates robust anti-tumor immunity as a monotherapy and in combination with checkpoint inhibitors

Abstract A member of the type-I IFN family, IFNα was also one of the first cytokines tested in the clinic as a therapy for cancer patients, leading to the approval of its use in chronic myelogenous leukemia, lymphoma, and malignant melanoma. Mechanistically, IFNα directly inhibits tumor cell proliferation and induces apoptosis, while simultaneously activating various innate and adaptive immune cell populations. Unfortunately, clinical use of IFNα has been hindered by the adverse events associated with its systemic administration, demonstrating the need for novel approaches to utilize this cytokine in the clinic. WTX-613 is a conditionally activated IFNα prodrug, or INDUKINE™ molecule, which has two identical half-life extension (HLE) domains tethered to IFNα2b via a proprietary tumor protease-sensitive linker. Following systemic administration, WTX-613 circulates as an inactive prodrug that is unable to bind to IFNα receptors in normal tissues due to the steric hinderance of the HLE domains complexed with albumin. However, when WTX-613 enters the tumor, proteases found in the tumor microenvironment (TME) cleave the protease-sensitive linkers, releasing fully active IFNα2b selectively in the tumor. Since human IFNα is not active in mice, a surrogate INDUKINE™ molecule containing mouse IFNα1 (mWTX-613/WW0610) was created. mWTX-613 treatment generated robust anti-tumor immunity in several murine syngeneic tumor models. In the MC38 model, mWTX-613 treatment was more efficacious than equimolar amounts of the recombinant cytokine (mIFNα1) and was accompanied by an increase in the therapeutic window. Mechanistically, systemic treatment with mWTX-613 resulted in prolonged peripheral exposure extending over a week beyond the time of the final dose. mWTX-613 treatment also resulted in a significant increase in the CD8/Treg ratio, and a robust increase in MHC I and MHC II expression by various professional antigen presenting cell populations. In addition, mWTX-613 treatment resulted in a clear and prolonged activation of both tumor infiltrating CD8+ T cells and NK cells. Transcriptionally, systemic treatment with mWTX-613 resulted in prolonged IFNα signaling within the TME, as demonstrated by significant enrichment in multiple Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with an anti-viral immune response. Finally, the combination of mWTX-613 treatment with various checkpoint inhibitor molecules resulted in improved anti-tumor efficacy in syngeneic models. Together, these data support the advancement of this innovative IFNα therapy into clinical testing. Citation Format: Christopher J. Nirschl, Heather R. Brodkin, Celesztina Domonkos, Kyriakos Economides, Daniel J. Hicklin, Nesreen Ismail, Cynthia Seidel-Dugan, Philipp Steiner, Zoe Steuert, William M. Winston, Andres Salmeron. WTX-613, (JZP898) a selectively activated IFNα INDUKINE™ molecule, reprograms the tumor microenvironment and generates robust anti-tumor immunity as a monotherapy and in combination with checkpoint inhibitors [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 1817.

Immune evasion of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2); molecular approaches.

In December 2019, a new betacoronavirus, known as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused an outbreak at the Wuhan seafood market in China. The disease was further named coronavirus disease 2019 (COVID-19). In March 2020, the World Health Organization (WHO) announced the disease to be a pandemic, as more cases were reported globally. SARS-CoV-2, like many other viruses, employs diverse strategies to elude the host immune response and/or counter immune responses. The infection outcome mainly depends on interactions between the virus and the host immune system. Inhibiting IFN production, blocking IFN signaling, enhancing IFN resistance, and hijacking the host's translation machinery to expedite the production of viral proteins are among the main immune evasion mechanisms of SARS-CoV-2. SARS-CoV-2 also downregulates the expression of MHC-I on infected cells, which is an additional immune-evasion mechanism of this virus. Moreover, antigenic modifications to the spike (S) protein, such as deletions, insertions, and also substitutions are essential for resistance to SARS-CoV-2 neutralizing antibodies. This review assesses the interaction between SARS-CoV-2 and host immune response and cellular and molecular approaches used by SARS-CoV-2 for immune evasion. Understanding the mechanisms of SARS-CoV-2 immune evasion is essential since it can improve the development of novel antiviral treatment options as well as vaccination methods.