Low IFN Research Articles

Abstract B019: Identification of gene signatures predictive of response to Ad-IFNα/Syn3 in bladder cancer

Abstract Introduction: Interferon α gene therapy (Ad-IFNα/Syn3) is FDA approved for treatment of BCG-unresponsive non-muscle invasive bladder cancer. Ad-IFNα/Syn3 is a non-replicating adenovirus that when transduced into target tissues, produces IFNa2b protein with anti-tumor activity. In the phase 3 multicenter trial, 45.5% of patients with carcinoma-in-situ and 60% of those with Ta/T1 disease who had a complete response to Ad-IFNα/Syn3 at 3 months remained recurrence free at 12 months. Targeting mechanisms of resistance and identifying predictive biomarkers to improve patient selection and overall response rates is a top priority. Towards this, we treated 29 human bladder cancer cell lines with Ad-IFNα/Syn3 and assessed their responses to therapy. We identified sensitive and resistant cell lines and gene signatures predictive of response. Methods: Human bladder cancer cell lines were grown in MEM supplemented with 10% FBS in 96-well plate. After overnight attachment, these were treated with Ad-IFNα/Syn3 (MOI 1000) and cell titre glow assay was performed 72h post-treatment. Cells were also seeded in 6-well plates. and after 24h cell-free supernatants were collected for ELISA and cells were collected for RNA isolation using RNAqueous Total RNA Isolation kit (Thermo Fisher). RNA quality was assessed using Tapestation. RNAseq analysis was carried out using Ion proton Sequencer. Ingenuity pathway analysis (IPA) and gene set enrichment were performed on the sequencing data for the cell lines. ELISA was used to measure IFNα and IL6 protein levels in cell free supernatants. We also tested organoids developed from bladder cancer patients to test drug efficacy and identify gene signatures. Results: Out of the 29 cell lines, we identified that sensitivity to Ad-IFNα/Syn3 was pronounced in luminal cells (8/13 luminal cell lines). Most basal cells were either resistant (>70% viability) or only moderately sensitive to Ad-IFNα/Syn3 (between 70-40% viability) when compared to control. We selected 7 cell lines for sequencing and ELISA measurements: luminal cell lines BV, UC6, Rt4V6 and UC5, and basal cell lines ScaBER, HT1197, and T24. All cell lines mentioned above could be successfully transduced by the adenoviral vector as measured by increased expression of IFNα protein in the cell-free supernatants. Ad-IFNα/Syn3-sensitive luminal cell lines at baseline had lower IFNα and IFNγ scores when compared to resistant basal cell lines. We identified several cell-death pathways enriched in treated cells that were sensitive to Ad-IFNα/Syn3. IL6 measurements in cell free supernatants revealed increased expression of IL6 after treatment in sensitive cells. However, in resistant cells IL6 expression was either high at baseline (ScaBER) or both undetectable at baseline and after treatment (UC5). Conclusion: Our results suggest that the luminal cell lines are more sensitive to Ad-IFNα/Syn3 and that high baseline expression of IFNα and IFNγ in basal cell lines contributes to resistance. IL6 levels are also predictive of response to Ad-IFNα/Syn3. Citation Format: Morgan E. Pierce, Alexis R. Steinmetz, Aruna Nannapaneni, Tanner S. Miest, David J. McConkey, Sharada Mokkapati, Colin P.N. Dinney. Identification of gene signatures predictive of response to Ad-IFNα/Syn3 in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B019.

Abstract CT127: A macrophages-based immunotherapy of solid tumors microenvironment: Preliminary results of the TEM-GBM_01 study

Abstract Glioblastoma multiforme (GBM) is an incurable tumor affecting the central nervous system. The reported median survival in the unfavorable patients′ subgroup with unmethylated MGMT promoter is 12.7 months with less than the 15% of patient surviving up to 2–years. Despite immunotherapies being able to slow or eradicate numerous tumors, none so far have extended survival in GBM. Using genetically modified autologous CD34+ HSPCs, we developed an advance medicinal product, (Temferon) designed to deliver interferon–α2 within the TME by Tie–2 expressing macrophages (TEMs). Temferon is currently in the clinic in a Phase 1/2a dose-escalation non-randomized open label study in newly diagnosed GBM with unmethylated MGMT. The study aims to evaluate the short– (up to 90 days) and long– (up to 2 years) term tolerability and safety of 5 escalating doses in up to 27 GBM patients. Following a sub–myeloablative conditioning regimen (Thiotepa + BCNU or Thiotepa + Busulfan or Busulfan alone), up to 3 million Temferon cells/kg are administered with a fixed dose of non–manipulated CD34+ supporter cells As of 27th July 2023, 21 GBM patients in 7 cohorts received incremental Temferon doses and 5 patients were still alive with a 17 months median OS and a 8.3 months median PFS (95% CI) post surgery. To date, no DLTs have been identified. In all patients, rapid engraftment and fast hematological recovery have been observed within 2 weeks from the transplantation. Grade 4 or 5 serious adverse events (n=7 patients), were mostly attributed to the conditioning chemotherapy, but steroid usage or disease progression were also listed. 1 SUSAR (persistent GGT elevation) occurred. The higher transduced cells proportion in the BM was observed 30 days after Temferon with a chimerism for the highest dose tested up to 40%, still detectable in the long–term. Very low IFNα concentrations were detected in the plasma, indicating a tight vector expression regulation. 7 patients underwent second surgery and genetically engineered cells were detected (in 85%) within the bulk tumor lesion (>3% in 3 patients). IFN–responsive genes (IGS) were up–regulated, suggesting a local IFNα release by TEMs. In 1 patient, a stable lesion, compared to a synchronous, progressing lesion, had a higher proportion of T cells & TEMs, an increased IGS e and myeloid re–programming. TCR blood and tumor sample sequencing showed a post–treatment increase of peripherally expanded clones frequency in the tumor samples, as evidence of a crosstalk between peripheral and tumor T cell repertoires . These data corroborate the initial evidence on safety and tolerability of Temferon and suggest that Temferon has potential to counteract disease progression in patients affected by unmethylated MGMT GBM. Clinical Trial Registry (if applicable): NCT03866109 Citation Format: Francesca Farina, Bernhard Gentner, Gaetano Finocchiaro, Marica Eoli, Alessia Capotondo, Elena Anghileri, Matteo Barcella, Valentina Brambilla, Matteo Carrabba, Valeria Cuccarini, Quintino Giorgio D'alessandris, Francesco DiMeco, Valeria Ferla, Paolo Ferroli, Filippo Gagliardi, Federico Legnani, Stefania Mazzoleni, Alessandro Olivi, Roberto Pallini, Marco Saini, Luigi Naldini, Carlo Russo, Fabio Ciceri. A macrophages-based immunotherapy of solid tumors microenvironment: Preliminary results of the TEM-GBM_01 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT127.

Bexmarilimab Activates Human Tumor-Associated Macrophages to Support Adaptive Immune Responses in Interferon-Poor Immune Microenvironments.

Immune checkpoint inhibitors (ICI) show substantially greater efficacy in inflamed tumors characterized by preexisting T-cell infiltration and IFN signaling than in noninflamed "cold" tumors, which often remain immunotherapy resistant. The cancer immunotherapy bexmarilimab, which inhibits the scavenger receptor Clever-1 to release macrophage immunosuppression and activate adaptive immunity, has shown treatment benefit in subsets of patients with advanced solid malignancies. However, the mechanisms that determine bexmarilimab therapy outcome in individual patients are unknown. Here we characterized bexmarilimab response in ovarian cancer ascites macrophages ex vivo using single-cell RNA sequencing and demonstrated increased IFN signaling and CXCL10 secretion following bexmarilimab treatment. We further showed that bexmarilimab was most efficacious in macrophages with low baseline IFN signaling, as chronic IFNγ priming abolished bexmarilimab-induced TNFα release. These results highlight an approach to target immunologically cold tumors and to increase the likelihood of their subsequent response to ICIs.

Association of Inflammatory Cytokine Levels with Extra Glandular Manifestations, Fatigue, and Disease Activity in Primary Sjögren's Syndrome in Saudi Patients: A Cross-Sectional Study.

Primary Sjögren's syndrome (pSS) is an autoimmune disease that can cause fatigue and extraglandular manifestations (EGMs). pSS is associated with cytokine network dysregulation, which may be related to the immune-mediated destruction of exocrine glands. We determined cytokine levels and their relationship to EGMs, the European League Against Rheumatism (EULAR) Sjögren's syndrome disease activity index (ESSDAI), and fatigue in Saudi patients with pSS. This study was a cross-sectional, single-center study. We included forty-one patients and 71 controls. Serum samples were collected from random healthy people and pSS patients who were followed in the rheumatology and pulmonary clinics of King Saud University Medical City in Riyadh, Saudi Arabia. Levels of the frequently studied cytokines were measured using Luminex xMAP technology. Each ESSDAI score and EGM were recorded, and the Arabic version of the fatigue severity scale (FSS) was applied to assess fatigue. The main outcome measures were cytokine levels in pSS Saudi patients using/not using immune-suppressive medications (ISMs). Thirty-six (87.8%) patients had one or more EGMs, and the mean ESSDAI score was 9.95 ± 7.73. There was a significant decrease in TNFα and IL-21 levels in the pSS group compared to those in the control group (p = 0.034 and p < 0.001, respectively), whereas IL-12 levels were significantly elevated in the pSS group (p = 0.002). Cytokine levels in patients who used ISMs were the same as those in patients who did not use medications. Decreased IL-1β (p = 0.014), IL-2 (p = 0.035), IL-6 (p = 0.014), and IL-35 (p = 0.010) levels were observed in patients who had EGMs. Patients who had low disease activity exhibited low IL-10 (p = 0.018) and high IFN-α (p = 0.049), IFN-β (p = 0.049), IL-1β (p = 0.006), and IL-35 (p = 0.032) levels compared to patients with high disease activity. A negative association between a positive fatigue score and IL-1β (p = 0.010), IL-2 (p = 0.037), IFN-α (p = 0.025), TNFα (p = 0.030), IL-17 (p = 0.029), IL-12 (p = 0.046), and IL-21 (p = 0.005) levels was found. Cytokine profiles correlate with EGMs, ESSDAI, and fatigue. Patients with controlled disease activity have a normal cytokine profile that is similar to that of controls.

Plasma Derived Extracellular Vesicles Modulate Monocyte IL-18, IFN-α and IL-10 responses in Adults with HIV-related Cognitive Impairment

Abstract Background: HIV-associated neurocognitive dysfunction persists despite suppressive antiretroviral therapy (ART). Extracellular vesicles (EVs) are emerging modulators of immunological responses. We have previously shown that EVs expressing monocyte activation and neuronal markers associated with HIV associated cognitive impairment (CI). Whether these EV changes observed in people with HIV (PWH) influence innate cellular function among those with CI remains unclear. Method: Purified EV samples from 58 HIV-infected individuals on ART with either cognitive impairment (CI, n=48) or normal cognition (NC, n=10) and 8 uninfected controls were isolated using differential centrifugation and co-cultured with PBMCs from a single uninfected donor for 18 hrs. Eight intracellular cytokines and 13 surface chemokines and myeloid differentiation markers were assessed by spectral flow cytometry. Non-parametric statistical T tests were used. Result: Monocytes (mono) showed differential effects on multiple cytokine responses and cell surface receptors after exposure to EVs from HIV-infected individuals with or without CI. Levels of mono IL-18 were significantly lower after incubation with EVs derived from HIV-infected individuals compared to uninfected controls. Based on cognitive status, EVs from HIV-CI group significantly lower % of IL-18 mono responses and lower IFN-α and higher IL-10 responses on a per cell basis when compared to the NC group. Conclusion: These results suggest EVs can modulate mono function irrespective of HIV status. PWH with CI exhibited altered EV driven mono cytokine responses. Our data support interventions that target EVs to reverse or restore mono perturbations and limit neuropathology in PWH on ART. Supported by grants from NIH (R21NS106970 and R01NS117458)

Abstract P3-06-08: Turning tumor cells into antigen-presenting cells for cancer immunotherapy

Abstract Miguel Lopez-Lago1, Sagarika pachhal1, Charles L. Wiseman1, William V. Williams1. 1BriaCell Therapeutics Corporation, Philadelphia, PA Turning tumor cells into antigen-presenting cells for cancer immunotherapy Background: Therapeutic cancer vaccines are based on specific stimulation of the immune system using tumor antigens to elicit an antitumor response. We have been conducting clinical trials using the breast cancer cell line SV-BR-1-GM as a therapeutic vaccine. SV-BR-1-GM is a GM-CSF expressing breast cancer cell line with features of an antigen presenting cell (APC) owning to the expression of several immunomodulatory molecules, including MHC-I (HLA-A, B &amp; C) and MHC-II (HLA-DRB3 &amp; -DRA). Initial results in patients treated with irradiated SV-BR-1-GM cells, low dose cyclophosphamide and local IFNα suggest that patients that match SV-BR-1-GM at least at 1 HLA allele are more likely to derive clinical benefit. This clinical observation, together with the fact that SV-BR-1-GM cells can directly activate CD4+ T-cells in an antigen-specific HLA-restricted manner, as demonstrated by an in vitro antigen presentation assay (1), lead us to hypothesize that SV-BR-1 (the parent cell line) can function as APC. To further enhance direct antigen presentation to T-cells, SV-BR-1 cells have been genetically modified to express co-stimulatory molecules, immunomodulatory cytokines, and an extended repertoire of HLA alleles. Methods: To generate an off-the-shelf semi-allogeneic cell therapy covering most of the population, SV-BR-1 was genetically modified to express an extended repertoire of HLA alleles. Based on population analysis, four cell lines, each carrying two (2) HLA-A and two (2) HLA-DRB3/4/5 alleles, should produce at least a single match in 99% of the population, with a 92% match at Class I HLA-A alleles and a 98% match at Class II HLA-DRB3/4/5 alleles. SV-BR-1 was genetically modified using CRISPR/cas9 deletion of the endogenous HLA-A and HLA-DRB3 alleles and subsequent lentiviral mediated expression of alternative HLA-A and DRB3 alleles. To generate tumor cell lines with enhanced direct antigen presentation to T-cells, SV-BR-1 cells were genetically modified to express co-stimulatory molecules and immunomodulatory cytokines by using a lentiviral mediated expression system. Results: Following sequential CRISPR/Cas9 editing, the SV-BR-1 cells were cloned, and one clone selected (clone 17) for further engineering. Lack of expression of HLA-A and HLA-DRB3 was confirmed using flow cytometry and DNA sequencing. Clone 17 was subsequently transduced with 6 lentiviral vectors each expressing 2 genes under the control of separate promoters:CD86-IL12, CD80-HLA-DRA, 4-1BBL-IL7, GM-CSF-IFN, HLA-allele-1-HLA-allele-2 and HLA-DR-allele-1-HLA-DR-allele-2. Four cell lines were generated with different combinations of HLA alleles. Following selection, cells were evaluated by ELISA, flow cytometry and RT-PCR to confirm gene expression. Cell lines that secreted GM-CSF, IFN IL12, IL7 and expressed CD80, CD86, 4-1BBL, and both Class I and Class II HLA alleles are then transferred to GMP manufacturing. These modified breast cancer cell lines will be used in clinical studies designed to first evaluate the safety of intradermal inoculation with the irradiated cells and later combined with other agents to augment the immune response. Each patient will be treated with the cell line(s) that match them at least at one HLA allele. 1. Lacher M. D. et al, Front Immunol. 2018 May 15;9:776 Citation Format: Miguel Lopez-Lago, William Williams, Charles Wiseman. Turning tumor cells into antigen-presenting cells for cancer immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-08.

CD30+ primary cutaneous tumoral adult T-cell leukemia/lymphoma successfully treated with zidovudine and interferon

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasia of mature peripheral T lymphocytes associated with human T-cell lymphotropic virus type 1 (HTLV-1). The primary cutaneous tumoral (PCT) type of adult T-cell leukemia/lymphoma presents as nodules and tumors without internal organ involvement, but with a bad prognosis. Here, we report a middle aged woman, who manifested a nodular lesion on the right leg. Histopathology showed moderately dense, lymphoid infiltrate of atypical, medium sized cells in the dermis and subcutaneous tissue. The cells were positive for CD3, CD4, CD30, and CD25, and negative for CD8, CD20, and anaplastic lymphoma kinase. The biopsy specimen showed a Ki-67 proliferation index of 90%. Serological testing for HTLV-1 was positive. The patient was diagnosed to have CD30+ PCT type ATL and was treated initially with localized radiation. The lesion regressed, however, there was a recurrence of the disease, which was treated with low dose interferon-alfa (IFN-α) and zidovudine, following which patient attained complete resolution and remained symptom free during the follow up period of 2 years. Our case highlights the usefulness of low dose IFN-α and zidovudine in the management of PCT type of CD30+ ATL.

The Porcine and Chicken Innate DNA Sensing cGAS-STING-IRF Signaling Axes Exhibit Differential Species Specificity.

The innate immune DNA sensing cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) signaling pathway plays a key role in host antiviral function. Although the cGAS-STING pathway has been extensively studied, the cGAS-STING signaling in livestock and poultry is not well understood, and whether the species specificity exists is still unknown. In this study, we found that porcine and chicken STING, but not cGAS, exhibit species differences in regulation of IFN; that is, porcine (p)STING mediates good induction of IFN in mammalian cells and low IFN induction in chicken DF-1 cells; on the contrary, chicken (ch)STING mediates IFN induction only in chicken cells but not in mammalian cells. Furthermore, it was found that the motifs pLxIS of pSTING and pLxVS of chSTING are responsible for the species disparity, with the IFN activity of pSTING and chSTING exchanged by swapping the two pLxI/VS motifs. The pLxI/VS motifs mediated the interactions of various STING with downstream IFN regulatory factors (IRFs), reflecting the species-specific pIRF3 and chIRF7. Next, the STING, IRFs, and STING-IRFs were reconstituted in porcine and chicken macrophages that were genetically knocked out for STING and/or IRFs by the CRISPR-Cas9 approach. The results showed that pSTING plus pIRF3 or chIRF7 are able to induce IFN; however, chSTING plus chIRF7 but not pIRF3 are able to induce IFN, suggesting that pIRF3 is specific and stringent, which underlies the inability of chSTING to induce IFN in mammalian cells. In summary, our findings reveal the differential species specificity in the cGAS-STING pathway and the underlying mechanisms, thus providing valuable insights on the cGAS-STING-IRF signaling axis for comparative immunology.

Interplay between B cell and GABA metabolism (GABAm) and association with immune evasion in breast carcinoma (BC).

1097 Background: GABAergic signaling has been reported to play a pivotal role in breast cancer (BC) tumorigenesis and metastasis, however, its role in immune modulation remains unclear. Recent in vitro and in vivo studies (Zhang et al., Nature, 2021) report the role of B cell-derived GABA metabolites in promoting anti-inflammatory macrophages (MM), thus limiting anti-tumor immunity. In this study, we aim to characterize the interplay between B cells and the GABAm pathway, as well as their associated immune infiltrates and cytokines. Methods: BC tumors (n = 9455) were analyzed by next generation sequencing (NextSeq, 592 Genes and WES, NovaSEQ) and whole transcriptome sequencing (WTS, NovaSeq) at Caris Life Sciences. Gene set variation analysis (GSVA) scores were used for GABAm pathway activity (GMPA). IFN score to test the likelihood of a tumor’s response to anti PD1 therapy and Immune cell fraction (quanTIseq) were assessed by mRNA analysis. Wilcoxon-Mann-Whitney test was applied (p without, q with multiple comparison correction). Correlation coefficients were calculated using spearman correlation. Results: GMPA demonstrated a statistically significant positive correlation with B cells fraction (r = 0.24, p &lt; 0.0001). When stratified by classical molecular subtypes, the positive correlations were exclusive to HR+ and HER2+ BC, and absent in TNBC. GMPA was the most enriched in HR+ BC, followed by HER2+ and TNBC. BC tumors with high B cell infiltration were then grouped into GMPA-high (B+G+, cutoff &gt; median for both) or GMPA-low (B+/G-), which likely represented tumors with B cell-derived high and low GMPA group, respectively. The GMPA-high group demonstrated significantly less fractions of MM1 (2.8 vs 3.7) and CD8+ T cells (0.8 vs 1.2) but greater MM2 (5.3 vs 4.9). mRNA levels of the MM2 marker IL10, a proposed marker of immune evasion, was significantly overexpressed in the B+/G+ group compared to the B+/G- group (fold change, FC = 1.39). mRNA levels of GAD1, a GABA-generating enzyme, were higher in B+/G+ than B+/G- (FC = 7.19). B+/G+ group had notably less IFN score than B+/G- group (-0.37 vs -0.27). When further stratified into molecular subtypes, concurrent more MM2 (5.4 vs 5.2) and less CD8+ T cell (0.74 vs 0.91) fractions were found in B+/G+ compared to B+/G- in HR+ tumors, but not in HER2+ or TNBC tumors. B+/G+ group also demonstrated a lower IFN score (-0.38 vs -0.32) in HR+ tumors. Additionally, IL10 and GAD1 were consistently overexpressed in B+/G+ regardless of subtype, reaching FC 7.9 in HR+ tumors. q &lt; 0.0001 for all comparisons. Conclusions: Our study is the largest clinical dataset to demonstrate the association of interplay between B cell and GABAm with immunogenicity. Our results support the potential role of B cell-derived GABAm metabolites in immune modulation in BC in a subtype-specific manner. Targeting small metabolites to modulate immune evasion in BC warrants further investigation.

Viral load is associated with mitochondrial dysfunction and altered monocyte phenotype in acute severe SARS-CoV-2 infection

Monocytes play a major role in the initial innate immune response to SARS-CoV-2. Although viral load may correlate with several clinical outcomes in COVID-19, much less is known regarding their impact on innate immune phenotype. We evaluated the monocyte phenotype and mitochondrial function in severe COVID-19 patients (n = 22) with different viral burden (determined by the median of viral load of the patients) at hospital admission. Severe COVID-19 patients presented lower frequency of CD14 + CD16- classical monocytes and CD39 expression on CD14 + monocytes, and higher frequency of CD14 + CD16 + intermediate and CD14-CD16 + nonclassical monocytes as compared to healthy controls independently of viral load. COVID-19 patients with high viral load exhibited increased GM-CSF, PGE-2 and lower IFN-α as compared to severe COVID-19 patients with low viral load (p < 0.05). CD14 + monocytes of COVID-19 patients with high viral load presented higher expression of PD-1 but lower HLA-DR on the cell surface than severe COVID-19 patients with low viral load. All COVID-19 patients presented decreased monocyte mitochondria membrane polarization, but high SARS-CoV-2 viral load was associated with increased mitochondrial reactive oxygen species. In this sense, higher viral load induces mitochondrial reactive oxygen species generation associated with exhaustion profile in CD14 + monocytes of severe COVID-19 patients. Altogether, these data shed light on new pathological mechanisms involving SARS-CoV-2 viral load on monocyte activation and mitochondrial function, which were associated with COVID-19 severity.

Cytokine gene polymorphisms of TNF, IFN-γ, and IL-12 as potential predictors in the onset of cervical disease in HR HPV-positive women with behavioral risk cofactors

Introduction/Objective. The aim of this study was to investigate the distribution of genotypes and alleles of proinflammatory cytokines TNF, IFN-?, and IL-12 and their effect on the development of a cervical illness and also to determine their associated influence with cofactors in HR HPV-positive women in Serbia. Methods. We have investigated 24 women and based on the cytological findings they were classified into four groups: PAP II, ASCUS, LSIL, and HSIL. Analysis of TNF, IL-12, and IFN-? polymorphisms was performed using the real-time PCR TaqMan method. Statistical analysis was performed using parametric and non-parametric tests and correlation and multiple regression analysis. Results. Significantly higher frequency of high production-related TNF AA genotype was observed in severe dysplasia. The correlation between TNF gene polymorphism and cervical findings were highly significant. There was a moderate, significant correlation between low production IFN-? AA genotype and earlier cervical infections. There was a significant correlation between the IL-12 polymorphism of the low production IL-12 AA genotype and cervical lesions. Conclusion. Results of this study show that HSIL is associated with significantly higher frequency of high production TNF AA genotype. It is known that polymorphisms of certain cytokine genes encoding proteins involved in Th1 and Th2 cellular responses may be associated with better or worse prognosis of cervical disease in women with persistent HR HPV infection. Therefore, they may be considered as biomarkers that may have a predictive role in the development of cervical cancer.

STEM-24. INTERFERON SIGNALING REGULATES THE PROLIFERATION AND MESENCHYMAL PHENOTYPE OF GLIOBLASTOMA STEM CELLS

Abstract Interferon (IFN) signaling contributes to stemness, cell proliferation, cell death, and cytokine signaling in cancer and immune cells; however, the role of IFN signaling in glioblastoma (GBM) and GBM stem-like cells (GSCs) is unclear. This study aimed to investigate the cancer cell-intrinsic IFN signaling in tumorigenesis and malignant phenotype of GBM. We characterized cell-intrinsic IFN signaling in The Cancer Genome Atlas, patient-derived cohorts of GSCs, and published single-cell RNA sequencing datasets by in-silico analyses. The in-silico findings were further validated by evaluating the cytokine secretion and using pharmacological activators and blockers of IFN/transducer and activator of transcription 1 (STAT1) signaling. We found that GSCs and GBM tumors exhibited differential cell-intrinsic IFN signaling, and high IFN/STAT1 signaling is associated with mesenchymal phenotype and poor survival outcomes. Ruxolitinib, a pharmacological inhibitor of IFN/STAT1, abolished the IFN/STAT1 signaling in GSCs with intrinsically high IFN signaling. IFN-γ treatment for 1 week promotes the mesenchymal phenotype in GSCs with low IFN signature. In addition, chronic inhibition of IFN/STAT1 signaling with ruxolitinib decreased cell proliferation and mesenchymal signatures (CD44, YKL40, and TIMP1) in GSCs with intrinsically active IFN/STAT1 signaling. Publicly available human glioma single-cell RNA-seq (scRNA-seq) datasets analyses showed that both tumor and nontumor cells expressed IFN signaling genes, and the mesenchymal signature was highly expressed in the same cluster where IFN signaling genes were upregulated. We demonstrated that cell-intrinsic IFN signaling in GSCs and GBM tumors is associated with mesenchymal signatures and cell proliferation. Our study provides evidence for the possibility of targeting IFN signaling in a specific group of GBM patients.

Serum biomarker profile orchestrating the seroconversion status of patients with autoimmune diseases upon planned primary 17DD Yellow fever vaccination

The present study aimed to investigate whether the serum biomarkers of immune response orchestrate the seroconversion status in patients with autoimmune diseases (AID) upon planned primary 17DD-YF vaccination. For this purpose a total of 161 individuals were enrolled in a prospective study, including patients with Rheumatoid Arthritis (RA = 38), Spondyloarthritis (SpA = 51), Systemic Lupus Erythematosus (SLE = 21) and Sjögren’s Syndrome (SS = 30) along with a group of healthy controls (HC = 21). Analysis of plaque reduction neutralization test (PRNT) titers and seropositivity rates along with the 17DD-YF viremia and serum biomarkers were carried out at distinct time points (D0/D3–4/D5–6/D7/D14–28). The results demonstrated an overall lower PRNT titer and seropositivity rate (170 vs. 448; 77 vs. 95%) in AID as compared to HC, especially in SpA and SLE subgroups. No significant differences were observed in the viremia levels amongst groups. In general, a more prominent serum biomarker response was observed in AID as compared to HC, throughout the timeline kinetics. Remarkably, AID/PRNT(−) exhibited higher levels of several biomarkers at baseline as compared to AID/PRNT+. Moreover, while AID/PRNT(+) exhibited earlier increase in serum biomarkers at D3–4/D5–6, the AID/PRNT(−) displayed higher response at later time points (D7/D14–D28). Of note, a synchronic increase of IFN-γ at the peak of viremia (D5–6) was observed in HC and AID/PRNT(+) groups, whereas a later asynchronous IFN-γ response was reported for AID/PRNT(−) at D7. The biomarker profile tends to deflate at post-vaccination timeline, highlighting a putative immunomodulatory effect of live attenuated 17DD-YF vaccine in AID/PRNT(+), but not in AID/PRNT(−). Altogether these data suggested that inflammatory status prior vaccination, low IFN-γ at viremia peak and the occurrence of asynchronous biomarker storm after 17DD-YF vaccination may orchestrate the lack of neutralizing antibody response γ.

Interleukin-3 is a predictive marker for severity and outcome during SARS-CoV-2 infections

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide health threat. In a prospective multicentric study, we identify IL-3 as an independent prognostic marker for the outcome during SARS-CoV-2 infections. Specifically, low plasma IL-3 levels is associated with increased severity, viral load, and mortality during SARS-CoV-2 infections. Patients with severe COVID-19 exhibit also reduced circulating plasmacytoid dendritic cells (pDCs) and low plasma IFNα and IFNλ levels when compared to non-severe COVID-19 patients. In a mouse model of pulmonary HSV-1 infection, treatment with recombinant IL-3 reduces viral load and mortality. Mechanistically, IL-3 increases innate antiviral immunity by promoting the recruitment of circulating pDCs into the airways by stimulating CXCL12 secretion from pulmonary CD123+ epithelial cells, both, in mice and in COVID-19 negative patients exhibiting pulmonary diseases. This study identifies IL-3 as a predictive disease marker for SARS-CoV-2 infections and as a potential therapeutic target for pulmunory viral infections.

Bioinformatic Approaches for Identification of Potential Repurposable Drugs in COVID-19

Introduction: Repurposing existing drugs approved for other conditions is crucial to identifying specific therapeutics against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing COVID-19 (coronavirus disease 2019) pandemic. Towards this attempt, it is important to understand how this virus hijacks the host system during the course of infection and determine potential virus- and host-targeted inhibitors.&#x0D; Methods: This study elucidates the underlying virus-host interaction based on differentially expressed gene profiling, functional enrichment and pathway analysis, protein-protein and protein-drug interactions utilizing the information on transcriptional response to SARS-CoV-2 infection from GSE147507 dataset containing COVID-19 case relative to healthy control and infected cell culture compared to uninfected one.&#x0D; Results: Low IFN signaling, chemokines level elevation, and proinflammatory cytokines release were observed markedly. We identified MYC-rapamycin and ABCG2-rapamycin interactions, and unique gene signatures in case (regulation of protein modification and MAPK signaling) as well as in cell (metabolic dysregulation and interferon signaling) different from known COVID-19 genes.&#x0D; Conclusion: Among a plethora of repurposable drugs those appearing here with unique gene signatures might be helpful in COVID-19&#x0D; Keywords: COVID-19, SARS-CoV-2, GSE 147507 dataset, protein-protein interaction, gene-drug interaction, repurposable drugs.

Whole transcriptome analysis of high and low IFN-α producers reveals differential response patterns following rhinovirus stimulation.

ObjectivesViral respiratory infections cause considerable morbidity and economic loss. While rhinoviruses (RV) typically cause little more than the common cold, they can produce severe infections and disease exacerbations in susceptible individuals, such as those with asthma. Variations in the regulation of key antiviral cytokines, particularly type I interferon (IFN‐α and IFN‐β), may contribute to RV susceptibility. To understand this variability, we compared the transcriptomes of high and low type I IFN producers.MethodsBlood mononuclear cells from 238 individuals with or without asthma were cultured in the presence or absence of RV. Those samples demonstrating high or low RV‐stimulated IFN‐α production (N = 75) underwent RNA‐sequencing.ResultsGene expression patterns were similar in samples from healthy participants and those with asthma. At baseline, the high IFN‐α producer group showed higher expression of genes associated with plasmacytoid dendritic cells, the innate immune response and vitamin D activation, but lower expression of oxidative stress pathways than the low IFN‐α producer group. After RV stimulation, the high IFN‐α producer group showed higher expression of genes found in immune response biological pathways and lower expression of genes linked to developmental and catabolic processes when compared to the low IFN‐α producer group.ConclusionsThese differences suggest that the high IFN‐α group has a higher level of immune system readiness, resulting in a more intense and perhaps more focussed pathogen‐specific immune response. These results contribute to a better understanding of the variability in type I IFN production between individuals.

Decreased Type I Interferon Production by Plasmacytoid Dendritic Cells Contributes to Severe Dengue.

The clinical presentation of dengue virus (DENV) infection is variable. Severe complications mainly result from exacerbated immune responses. Type I interferons (IFN-I) are important in antiviral responses and form a crucial link between innate and adaptive immunity. Their contribution to host defense during DENV infection remains under-studied, as direct quantification of IFN-I is challenging. We combined ultra-sensitive single-molecule array (Simoa) digital ELISA with IFN-I gene expression to elucidate the role of IFN-I in a well-characterized cohort of hospitalized Cambodian children undergoing acute DENV infection. Higher concentrations of type I IFN proteins were observed in blood of DENV patients, compared to healthy donors, and correlated with viral load. Stratifying patients for disease severity, we found a decreased expression of IFN-I in patients with a more severe clinical outcome, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). This was seen in parallel to a correlation between low IFNα protein concentrations and decreased platelet counts. Type I IFNs concentrations were correlated to frequencies of plasmacytoid DCs, not DENV-infected myloid DCs and correlated inversely with neutralizing anti-DENV antibody titers. Hence, type I IFN produced in the acute phase of infection is associated with less severe outcome of dengue disease.

Similarities and differences between the \u2018cytokine storms\u2019 in acute dengue and COVID-19

Severe pneumonia and multiorgan dysfunction in COVID-19 and dengue haemorrhagic fever (DHF) are two diseases that can associate with an altered immune response to the infecting virus. To determine the similarities and differences in the cytokine and chemokine responses in these two infections, we compared responses in patients with varying severity of COVID-19 and acute dengue at different time points of illness. During early disease, patients who proceeded to develop COVID-19 severe pneumonia (SP) and DHF had significantly higher levels of IL-6, IL-10 and MIP3α than those who developed mild illness. The lowest levels of IFNγ in early illness were seen in those who succumbed to their illness due to COVID-19. Levels of serum IL-10 (p = 0.0001), IL-6 (p = 0.002), MIP-3α (p = 0.02) and CD40-L levels (p = 0.002) significantly increased from 5 to 9 day of illness to 10–21 day of illness in patients with moderate-to-severe COVID-19, but not in those with mild illness. In contrast, these cytokine/chemokine levels remained unchanged in those with DHF or dengue fever (DF) during febrile and critical phases. Although IL-10 levels were significantly higher in COVID-19 patients with SP, patients with DHF had 25-fold higher levels, whereas IL-6 levels were 11-fold higher in those with COVID-19 SP. IL-10 and other cytokines were evaluated in a larger cohort of patients during early illness (≤ 4 days) who proceeded to develop DF (n = 71) or DHF (n = 64). Of the cytokines evaluated, IL-10 was significantly higher (p < 0.0001) in those who went on to develop DHF compared to DF. Low IFNγ response to the SARS-CoV2 and high levels of immunosuppressive IL-10 in both COVID-19 and dengue during early illness are indicators of an altered antiviral response potentially contributing to disease severity.

GSK2245035, a TLR7 agonist, Does Not Increase Pregnancy Loss in Cynomolgus Monkeys

GSK2245035, a small molecule Toll-like Receptor 7 (TLR7) agonist developed for immunomodulatory treatment for allergic airways disease, aimed to reduce Th2 and enhance Th1/Treg responses to aeroallergens via the local induction of type I interferons (IFNs). GSK2245035 demonstrated selectivity for potent release of type I IFNs compared to TNF-α and IL-6, with dose dependent increases in the interferon inducible chemokine, IP-10, in the nasal compartment. Implantation and parturition require pro-inflammatory processes including IFNs, Interferon Stimulated Genes, TNFα and IP-10 while pregnancy requires immune regulation to maintain maternal fetal immune tolerance, and recombinant type I IFNs induced abortions in monkeys. Due to its mechanism of action, GSK2245035 was studied at pharmacologically and clinically relevant doses in a monkey pregnancy model. Monkeys received 0, 3 or 30 ng/kg/week GSK2245035 intranasally once weekly, from Day 20 postcoitum through Day 63 postpartum. Although systemic IFN-α and IP-10 levels were approximately 14.8 or 40 -fold (respectively) above predose levels at 3 or 30 ng/kg/week, respectively, there were no effects on pregnancy and infant outcome. Non-adverse effects included increased incidence of nasal discharge, increased maternal body temperature at 30 ng/kg/week and dose-dependent increases in maternal IP-10 and IFN-α and decreased infant anti-KLH IgM and IgG titers following KLH immunization at ≥3 ng/kg/week, relative to controls. Potentially, lower IFN-α and IP-10 levels as well as once-weekly intranasal dosing vs daily subcutaneous or intramuscular dosing with recombinant type I IFNs could explain the lack of pregnancy effects; however, there was an undesired impact on offspring immune function.

Value of blood IFN-I levels in COVID-19 management

Abstract Coronavirus disease 2019 (COVID-19) which is a respiratory system disease has created inevitable pandemic. In the course of disease; the uncontrolled inflammatory response may cause tissue damage. 80–90% of patients undergo mild-to-moderate disease symptoms, the rest of the patients proceeds to critical or severe disease. Type I Interferons (IFN–I) are crucial immune mediators in order to host responses to viral infection likewise those are major components of the innate immune system moreover serve as critical antiviral molecules. IFN-I are within the first cytokines secreted during a viral infection. Until now, IFN-I response has been evaluated in patients with COVID-19 in a few publications and its contribution to the viral load control and inflammation is very little known. In those studies, the researchers have found that IFN-I deficiency which characterized by no IFN- β and low IFN- α production and activity is a hallmark of severe and critical COVID-19. Until recently, measurement of circulating serum levels of IFN-α have been limited owing to the limits of conventional immunoassay technology. Limit of detection problems of immunoassays has started to figure out thanks to SIMOA. On the top of that, generally the platforms that use SIMOA technology is 10–100 fold more sensitive than most conventional immunoassays. We should strive to do research that includes IFN-I with using platforms that include SIMOA technology, especially in severe COVID-19.