The pulmonary immune system consists of a network of tissue-resident cells as well as immune cells that are recruited to the lungs during infection and/or inflammation. How these immune components communicate during an acute poxvirus infection is not well understood. Intranasal infection of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we provide evidence that type II alveolar epithelial cells (AECIIs) function as the sentinels of pulmonary infection of vaccinia virus by inducing IFN-β and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic acid-sensing pathways and the type I IFN positive feedback loop. This leads to the recruitment and activation of CCR2+ inflammatory monocytes in the infected lungs and their differentiation into Lyve1- interstitial macrophages (Lyve1- IMs), which efficiently engulf viral particles and block viral replication. Our results provide novel insights into how innate immune-sensing of viral infection by lung AECIIs influences the activation and differentiation of CCR2+ inflammatory monocytes to defend pulmonary poxvirus infection.
Multiple treatment modalities for Kaposi sarcoma (KS) have been reported, including chemotherapy, radiation therapy, surgical excision, electrochemotherapy, and cryotherapy. Common topical treatments include timolol, imiquimod, and alitretinoin. We searched our institutional database for patients with ICD‐9 or 10 codes for KS seen by a dermatologist with experience in KS management from July 1, 2004 to January 1, 2022. We screened patient charts to include patients who received combination therapy of cryotherapy followed by topical imiquimod three times a week for 2 months (n = 9). Patients were followed in the clinic every 3 months. Time to resolution was assessed by photographic evidence of resolution as determined by a dermatologist and corroborated with clinical documentation in patient charts. Median age (IQR) at KS diagnosis was 58 (27.5) years. All patients were male (n = 9, 100%). Majority were white (n = 7, 78%) and non‐Hispanic (n = 8, 89%). Five (56%) had classic KS, one (11%) had HIV‐associated KS, and three (33%) were HIV‐negative men who have sex with men. Median time to resolution was 30.5 weeks, with a median of two treatments. In our study, 93% (n = 42/45) of lesions and 89% (n = 8/9) of patients experienced complete resolution during a median (range) duration of follow‐up of 58 (13–209) weeks. Side effects were limited to pain during cryotherapy, occasional blister formation after cryotherapy, and mild inflammation due to imiquimod. No infections were observed. Combination therapy of cryotherapy and topical imiquimod may be an efficacious and comparatively low‐risk treatment for limited, cutaneous KS.
Immune checkpoint blockade (ICB) has demonstrated clinical success in “inflamed” tumors with significant T-cell infiltrates, but tumors with an immune-desert tumor microenvironment (TME) fail to benefit. The tumor cell-intrinsic molecular mechanisms of the immune-desert phenotype remain poorly understood. Here, we demonstrate that inactivation of the Polycomb-repressive complex 2 (PRC2) core components, EED or SUZ12, a prevalent genetic event in malignant peripheral nerve sheath tumor (MPNST) and sporadically in other cancer types, drives a context-dependent immune-desert TME. PRC2 inactivation reprograms the chromatin landscape that leads to a cell-autonomous shift from primed baseline signaling-dependent cellular responses (e.g., interferon γ) to PRC2-regulated development and cellular differentiation transcriptional programs. Further, PRC2 inactivation reprograms the TME, leads to diminished tumor immune infiltrates and immune evasion through reduced chemokine production and impaired antigen presentation and T-cell priming, and confers ICB primary resistance through blunted T-cell recruitment in vivo. We demonstrate that strategies that enhancing innate immunity via intratumoral delivery of inactivated modified vaccinia virus Ankara (MVA) leads to increased tumor immune infiltrates and sensitizes PRC2-loss tumors to ICB. Our results provide novel molecular mechanisms of context-dependent dysfunctional epigenetic reprogramming that underline the immune-desert phenotype in MPNST and other cancers with PRC2 inactivation. Importantly, our findings highlight genetic-inactivation of PRC2 as a novel context-dependent ICB therapeutic resistance biomarker in cancer, and caution that therapeutic strategies that non-selectively target PRC2 in the host may lead to undesirable context-dependent immune evasion and ICB resistance in tumors. Our studies also point to intratumoral delivery of immunogenic therapeutic viruses as an initial strategy to modulate the immune-desert TME and capitalize on the clinical benefit of ICB. Citation Format: Juan Yan, Yuedan Chen, Amish J. Patel, Cindy J. Lee, Sarah Warda, Briana G. Nixon, Elissa W. P. Wong, Miguel A. Miranda-Román, Ning Yang, Yi Wang, Jessica Sher, Emily Giff, Fanying Tang, Ekta Khurana, Sam Singer, Yang Liu, Phillip M. Galbo, Jesper L. Maag, Richard P. Koche, Deyou Zheng, Cristina R. Antonescu, Ming Li, Liang Deng, Yu Chen, Ping Chi. Tumor-intrinsic PRC2 inactivation drives a context-dependent immune-desert tumor microenvironment [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 6164.
Vaccinia virus C7 protein is an important host-range factor for vaccinia virus life cycle in mammalian cells. C7L homologs are present in almost all of the poxviruses that infect mammalian hosts. Type I IFN plays an important role in host defense of viral infection, and yet, the role of C7 in immune modulation of the IFN pathway is unclear. We have previously reported that the highly attenuated modified vaccinia virus Ankara (MVA) infection of conventional dendritic cells (cDCs) induces type I IFN via the cGAS/STING/TBK1/IRF3 pathway. In this study, we find that ectopic C7 expression blocks STING, TBK1, or IRF3-induced IFNB and ISRE (interferon stimulated response element) promoter activation. Murine or human macrophage cell lines that overexpress C7 have blunted innate immune responses to DNA or RNA stimuli, or the infection of DNA or RNA viruses. Overexpression of C7 also attenuates ISG gene expression induced by IFN-b treatment. MVA with deletion of C7L (MVAΔC7L) infection of cDCs induces higher levels of type I IFN than MVA. C7 blocks IFN-b-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway via preventing Stat2 phosphorylation. C7 directly interacts with stat2 as demonstrated by co-immunoprecipitation studies. Taken together, our results provide evidence that C7 has dual inhibitory roles in type I IFN production and signaling.
The cytosolic DNA sensor cGAS plays an important role in detecting viral nucleic acid, which leads to type I IFN production. We have previously shown that infection with conventional dendritic cells with modified vaccinia virus Ankara (MVA), a highly attenuated vaccinia strain (VACV), induces IFN production via a cGAS/STING-dependent mechanism. However, MVA or VACV infection triggers cGAS degradation and its mechanism is still unknown. VACV is a cytoplasmic DNA virus, which encodes more than 200 genes. In this study, we screened 70 vaccinia viral early genes for inhibition of cGAS/STING pathway using a dual luciferase system. We found that vaccinia E5 is a dominant inhibitor of cGAS and is the key protein mediating cGAS degradation. MVAΔE5R induces much higher levels of type I IFN than MVA in multiple cell types, including bone marrow derived dendritic cells (BMDC), bone marrow-derived macrophages (BMDM), and skin primary fibroblasts. MVAΔE5R-mediated type I IFN production is dependent on cGAS. Furthermore, MVAΔE5R gains replication capability in cGAS−/− skin fibroblasts. As a vaccine vector, skin scarification or intradermal vaccination with MVAΔE5R-OVA leads to much higher OVA-specific CD8+ T cell responses than MVA-OVA in vivo. Intratumoral injection of MVAΔE5R leads to stronger anti-tumor immune responses and better survival compared with MVA. Finally, in an intranasal infection model, VACVΔE5R is at least 100-fold attenuated compared with WT VACV. Taken together, our results provide strong evidence that E5 is a key viral virulence factor targeting the cytosolic DNA sensor cGAS and thereby inhibits type I IFN production.
Vaccinia virus C7 protein is an important host-range factor for the vaccinia virus life cycle in mammalian cells. Type I IFN plays an important role in host defense of viral infection. However, the role of C7 in immune modulation of the IFN pathway is unclear. Here, we find that C7 functions as a dual inhibitor of IFN production and IFN signaling by interfering with the activation of two transcription factors IRF3 and STAT2. Furthermore, vaccinia virus with deletion of C7 (VACVΔC7L) is non-pathogenic in wild-type C57BL/6J mice in an intranasal infection model. We elucidated the immunological mechanisms underlying the attenuation phenotype of VACVΔC7L, which can be summarized as follows: (i) intranasal infection of VACVΔC7L triggers the production of interferons (IFNs), proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BAL), whereas WT VACV does not; (ii) intranasal infection of VACVΔC7L results in the recruitment of dendritic cells, inflammatory monocytes, neutrophils, CD8+, and CD4+ in the BAL, whereas WT VACV does not; and (iii) infection of primary type II lung alveolar epithelial cells (AECs) with VACVΔC7L induces IFN, proinflammatory cytokine and chemokine gene expression and protein secretion, whereas WT VACV does not. However, VACVΔC7L dramatically gained virulence in STAT2 or IFNAR1-deficient mice, with increased titers in the lungs and systemic dissemination of the viruses to the blood and distant organs. Taken together, our results provide evidence that vaccinia C7 is a key virulence factor that antagonize both IFN production and signaling and IFNAR1 and STAT2 are critical in restricting vaccinia viral replication in the lung alveolar epithelial cells.
Immune checkpoint blockade (ICB) has demonstrated clinical success in inflamed tumors with significant T-cell infiltrates, but tumors with an immune-desert tumor microenvironment (TME) fail to benefit. The tumor cell-intrinsic molecular mechanisms of the immune-desert phenotype remain poorly understood. Here, we demonstrate that inactivation of the Polycomb-repressive complex 2 (PRC2) core components, EED or SUZ12, a prevalent genetic event in malignant peripheral nerve sheath tumor (MPNST) and sporadically in other cancer types, drives a context-dependent immune-desert TME. PRC2 inactivation reprograms the chromatin landscape that leads to a cell-autonomous shift from primed baseline signaling-dependent cellular responses (e.g., interferon gamma) to PRC2-regulated development and cellular differentiation transcriptional programs. Further, PRC2 inactivation reprograms the TME, leads to diminished tumor immune infiltrates and immune evasion through reduced chemokine production and impaired antigen presentation and T-cell priming, and confers ICB primary resistance through blunted T-cell recruitment in vivo. We demonstrate that strategies that enhancing innate immunity via intratumoral delivery of inactivated modified vaccinia virus Ankara (MVA) leads to increased tumor immune infiltrates and sensitizes PRC2-loss tumors to ICB. Our results provide novel molecular mechanisms of context-dependent dysfunctional epigenetic reprogramming that underline the immune-desert phenotype in MPNST and other cancers with PRC2 inactivation. Importantly, our findings highlight genetic-inactivation of PRC2 as a novel context-dependent ICB therapeutic resistance biomarker in cancer, and caution that therapeutic strategies that non-selectively target PRC2 in the host may lead to undesirable context-dependent immune evasion and ICB resistance in tumors. Our studies also point to intratumoral delivery of immunogenic therapeutic viruses as an initial strategy to modulate the immune-desert TME and capitalize on the clinical benefit of ICB.
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