Host immune responses associated with SARS-CoV-2 Omicron infection result in protection or pathology during reinfection depending on mouse genetic background.

Rapid emergence of antigenic distinct SARS-CoV-2 variants implies a greater risk of reinfection as viruses can escape neutralizing antibodies induced by vaccination or previous viral exposure. Disease severity during COVID-19 depends on many variables such as age-related comorbidities, host immune status and genetic variation. The host immune response during infection with SARS-CoV-2 may contribute to disease severity, which can range from asymptomatic to severe with fatal outcome. Furthermore, the extent of host immune response activation may rely on underlying genetic predisposition for disease or protection. To address these questions, we performed immune profiling studies in mice with different genetic backgrounds - transgenic K18-hACE2 and wild-type 129S1 mice - subjected to reinfection with the severe disease-causing SARS-CoV-2 B.1.351 variant, 30 days after experimental milder BA.1 infection. BA.1 preinfection conferred protection against B.1.351-induced morbidity in K18-hACE2 mice but aggravated disease in 129S1 mice. We found that he cytokine/chemokine profile in B.1.351 re-infected 129S1mice is similar to that during severe SARS-CoV-2 infection in humans and is characterized by a much higher level of IL-10, IL-1ß, IL-18 and IFN-γ, whereas in B.1.351 re-infected K18-hACE2 mice, the cytokine profile echoes the signature of naïve mice undergoing viral infection for the first time. Interestingly, the enhanced pathology observed in 129S1 mice upon reinfection cannot be attributed to a less efficient induction of adaptive immune responses to the initial BA.1 infection, as both K18-hACE2 and 129S1 mice exhibited similar B and T cell responses at 30 DPI against BA.1, with similar anti-BA.1 or B.1.351 spike-specific ELISA binding titers, levels of germinal center B-cells, and SARS-CoV-2-Spike specific tissue-resident T-cells. Long-term effects of BA.1 infection are associated with differential transcriptional changes in bronchoalveolar lavage-derived CD11c + immune cells from K18-hACE2 and 129S1, with K18-hACE2 CD11c + cells showing a strong antiviral defense gene expression profile whereas 129S1 CD11c + cells showed a more pro-inflammatory response. In conclusion, initial infection with BA.1 induces cross-reactive adaptive immune responses in both K18-hACE2 and 129S1 mice, however the different disease outcome of reinfection seems to be driven by differential responses of CD11c + cells in the alveolar space.

Immune responses elicited by ssRNA(-) oncolytic viruses in the host and in the tumor microenvironment.

Oncolytic viruses (OVs) are at the forefront of biologicals for cancer treatment. They represent a diverse landscape of naturally occurring viral strains and genetically modified viruses that, either as single agents or as part of combination therapies, are being evaluated in preclinical and clinical settings. As the field gains momentum, the research on OVs has been shifting efforts to expand our understanding of the complex interplay between the virus, the tumor and the immune system, with the aim of rationally designing more efficient therapeutic interventions. Nowadays, the potential of an OV platform is no longer defined exclusively by the targeted replication and cancer cell killing capacities of the virus, but by its contribution as an immunostimulator, triggering the transformation of the immunosuppressive tumor microenvironment (TME) into a place where innate and adaptive immunity players can efficiently engage and lead the development of tumor-specific long-term memory responses. Here we review the immune mechanisms and host responses induced by ssRNA(-) (negative-sense single-stranded RNA) viruses as OV platforms. We focus on two ssRNA(-) OV candidates: Newcastle disease virus (NDV), an avian paramyxovirus with one of the longest histories of utilization as an OV, and influenza A (IAV) virus, a well-characterized human pathogen with extraordinary immunostimulatory capacities that is steadily advancing as an OV candidate through the development of recombinant IAV attenuated platforms.

Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity.

Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. The presence of damaging mutations in the SWI/SNF chromatin remodeling complex, such as the SMARCA4 (BRG1) catalytic subunit, has been associated with improved response to ICB, however the mechanism by which this occurs is unclear. The aim of this current study was to examine the alterations in tumor cell-intrinsic and extrinsic immune signaling caused by SMARCA4 loss. Using OC models with loss-of-function mutations in SMARCA4 , we found that SMARCA4 loss resulted in increased cancer cell-intrinsic immunogenicity, characterized by upregulation of long-terminal RNA repeats such as endogenous retroviruses, increased expression of interferon-stimulated genes, and upregulation of antigen presentation machinery. Notably, this response was dependent on IRF3 signaling, but was independent of the type I interferon receptor. Mice inoculated with cancer cells bearing SMARCA4 loss demonstrated increased activation of cytotoxic T cells and NK cells in the tumor microenvironment as well as increased infiltration with activated dendritic cells. These results were recapitulated when animals bearing SMARCA4- proficient tumors were treated with a BRG1 inhibitor, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to reverse immune evasion in OC.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Increased p53 expression induced by APR-246 reprograms tumor-associated macrophages to augment immune checkpoint blockade.

In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53-dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule-based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.

Avian Paramyxovirus 4 Antitumor Activity Leads to Complete Remissions and Long-term Protective Memory in Preclinical Melanoma and Colon Carcinoma Models.

Avulaviruses represent a diverse subfamily of non-segmented negative strand RNA viruses infecting avian species worldwide. To date, 22 different serotypes have been identified in a variety of avian hosts, including wild and domestic birds. APMV-1, also known as Newcastle disease virus (NDV), is the only avulavirus that has been extensively characterized due to its relevance for the poultry industry and, more recently, its inherent oncolytic activity and potential as a cancer therapeutic. An array of both naturally-occurring and recombinant APMV-1 strains has been tested in different preclinical models and clinical trials, highlighting NDV as a promising viral agent for human cancer therapy. To date, the oncolytic potential of other closely related avulaviruses remains unknown. Here, we have examined the in vivo anti-tumor capability of prototype strains of APMV serotypes -2, -3, -4, -6, -7, -8 and -9 in syngeneic murine colon carcinoma and melanoma tumor models. Our studies have identified APMV-4 Duck/Hong Kong/D3/1975 virus as a novel oncolytic agent with greater therapeutic potential than one of the NDV clinical candidate strains, La Sota. Intratumoral administration of the naturally-occurring APMV-4 virus significantly extends survival, promotes complete remission, and confers protection against re-challenge in both murine colon carcinoma and melanoma tumor models. Furthermore, we have designed a plasmid rescue strategy that allows us to develop recombinant APMV-4-based viruses. The infectious clone rAPMV-4 preserves the extraordinary antitumor capacity of its natural counterpart, paving the way to a promising next generation of viral therapeutics.

Tim-4+ cavity-resident macrophages impair anti-tumor CD8+ T cell immunity.

Immune checkpoint blockade (ICB) has been a remarkable clinical advance for cancer; however, the majority of patients do not respond to ICB therapy. We show that metastatic disease in the pleural and peritoneal cavities is associated with poor clinical outcomes after ICB therapy. Cavity-resident macrophages express high levels of Tim-4, a receptor for phosphatidylserine (PS), and this is associated with reduced numbers of CD8+ T cells with tumor-reactive features in pleural effusions and peritoneal ascites from patients with cancer. We mechanistically demonstrate that viable and cytotoxic anti-tumor CD8+ T cells upregulate PS and this renders them susceptible to sequestration away from tumor targets and proliferation suppression by Tim-4+ macrophages. Tim-4 blockade abrogates this sequestration and proliferation suppression and enhances anti-tumor efficacy in models of anti-PD-1 therapy and adoptive T cell therapy in mice. Thus, Tim-4+ cavity-resident macrophages limit the efficacy of immunotherapies in these microenvironments.

Unraveling tumor-immune heterogeneity in advanced ovarian cancer uncovers immunogenic effect of chemotherapy.

In metastatic cancer, the degree of heterogeneity of the tumor microenvironment (TME) and its molecular underpinnings remain largely unstudied. To characterize the tumor-immune interface at baseline and during neoadjuvant chemotherapy (NACT) in high-grade serous ovarian cancer (HGSOC), we performed immunogenomic analysis of treatment-naive and paired samples from before and after treatment with chemotherapy. In treatment-naive HGSOC, we found that immune-cell-excluded and inflammatory microenvironments coexist within the same individuals and within the same tumor sites, indicating ubiquitous variability in immune cell infiltration. Analysis of TME cell composition, DNA copy number, mutations and gene expression showed that immune cell exclusion was associated with amplification of Myc target genes and increased expression of canonical Wnt signaling in treatment-naive HGSOC. Following NACT, increased natural killer (NK) cell infiltration and oligoclonal expansion of T cells were detected. We demonstrate that the tumor-immune microenvironment of advanced HGSOC is intrinsically heterogeneous and that chemotherapy induces local immune activation, suggesting that chemotherapy can potentiate the immunogenicity of immune-excluded HGSOC tumors.

Preparation of single cells from tumors for single-cell RNA sequencing.

Intratumoral heterogeneity of cancer cells and tumor-infiltrating immune cells is increasingly being viewed as a key factor driving tumor progression and response to therapy. Over the past several years, technological advances have created powerful tools to analyze the tumor microenvironment on a single-cell level, including mass cytometry and single-cell RNA sequencing, which is particularly pertinent to tumor immunology and cancer immunotherapy. The integrity and reliability of the data generated from these single-cell technologies, however, are highly influenced by the process and quality of sample preparation, which, if carried out inappropriately, has a potential to produce misleading results. In this chapter, we describe a protocol for the generation of single cell suspensions from human tumor samples that has been optimized for single-cell RNA sequencing. This protocol can be easily adapted for other single cell applications such as mass and flow cytometry. Throughout the entire workflow, we aim to maximize viability and minimize factors contributing to cellular stress that could affect downstream analyses.

Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary.

Sclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic-phenotypic correlation in ovarian neoplasms.

Virus, Vessel, Victory: A Novel Approach to Tumor Killing.

Ovarian cancer is intimately dependent on aberrant tumor vasculature. A unique therapeutic approach combining a systemic oncolytic virus with a novel vascular-normalizing agent in ovarian cancer demonstrates the potential to enhance antitumor efficacy through improved delivery of the virus and enhanced infiltration of immune cells into the tumors.See related article by Matuszewska et al., p. 1624.