Type-1 IFN Research Articles

Association of serum interferon alpha-2a levels with disease severity and prognosis in systemic sclerosis.

To determine serum type I interferon IFN-α2a concentrations in systemic sclerosis (SSc) patients, explore its association with cytokine/chemokine expressions, and evaluate correlation with the phenotype including the predictive value for interstitial lung disease (ILD) progression. Serum samples were obtained from 200 SSc patients and 29 healthy controls. IFN-α2a levels were measured by ultrasensitive electrochemiluminescence assay. Pro-inflammatory and chemokine panels were determined by Luminex® Discovery Assay multiplex kit. Baseline SSc disease characteristics were recorded together with longitudinal data for determining ILD progression after 2 years. IFN-α2a concentrations were higher in SSc patients compared with controls, although not reaching significance (means ± SD of 49.20 ± 156.8 pg/ml vs 9.606 ± 4.399 pg/ml, respectively; (p = 0.158)). Using the cut-off of 15.9 pg/ml, we identified 62 patients as having a type 1 (T1) IFN signature in their circulation. Patients with an IFN signature had significantly higher levels of chemokines (CCL8, CCL19, CXCL10, CXCL11), and the cytokine IL-1α compared with those without an IFN signature. IFN-α2a concentrations strongly correlated with a T1 IFN related chemokine score supporting activation of this pathway. Phenotyping association queries revealed association between IFN values and both skin and ILD involvements at baseline. Longitudinal data did not identify IFN as a predictive marker for ILD progression. Using serum determinations, the activation of the T1 IFN pathway showed strong correlations with inflammatory mediators and associations with clinical manifestations, especially skin fibrosis and ILD in SSc patients. However, activated IFN pathway was not predictive of ILD progression.

Downregulation of ABLIM3 confers to the metastasis of neuroblastoma via regulating the cell adhesion molecules pathway

Neuroblastoma (NB) is the most prevalent extracranial solid tumor in pediatric patients, and its treatment failure often associated with metastasis. In this study, LASSO, SVM-RFE, and random forest tree algorithms, was used to identify the pivotal gene involved in NB metastasis. NB cell lines (SK-N-AS and SK-N-BE2), in conjunction with NB tissue were used for further study. ABLIM3 was identified as the hub gene and can be an independent prognostic factor for patients with NB. The immunohistochemical analysis revealed that ABLIM3 is negatively correlated with the metastasis of NB. Patients with low expression of ABLIM3 had a poor prognosis. High ABLIM3 expression correlated with APC co-stimulation and Type1 IFN response, and TIDE analysis indicated that patients with low ABLIM3 expression exhibited enhanced responses to immunotherapy. Downregulation of ABLIM3 by shRNA transfection increased the migration and invasion ability of NB cells. Gene Set Enrichment Analysis (GSEA) revealed that genes associated with ABLIM3 were primarily enriched in the cell adhesion molecules (CAMs) pathway. RT-qPCR and western blot analyses demonstrated that downregulation of ABLIM3 led to decreased expression of ITGA3, ITGA8, and KRT19, the key components of CAMs. This study indicated that ABLIM3 can be an independent prognostic factor for NB patients, and CAMs may mediate the effect of ABLIM3 on the metastasis of NB, suggesting that ABLIM3 is a potential therapeutic target for NB metastasis, which provides a novel strategy for future research and treatment strategies for NB patients.

Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

The Chikungunya virus (CHIKV) causes Chikungunya fever, a disease characterized by symptoms such as arthralgia/polyarthralgia. Currently, there are no antivirals approved against CHIKV, emphasizing the need to develop novel therapies. The imidazonaphthyridine compound (RO8191), an interferon-α (IFN-α) agonist, was reported as a potent inhibitor of HCV. Here RO8191 was investigated for its potential to inhibit CHIKV replication in vitro. RO8191 inhibited CHIKV infection in BHK-21 and Vero-E6 cells with a selectivity index (SI) of 12.3 and 37.3, respectively. Additionally, RO8191 was capable to protect cells against CHIKV infection, inhibit entry by virucidal activity, and strongly impair post-entry steps of viral replication. An effect of RO8191 on CHIKV replication was demonstrated in BHK-21 through type-1 IFN production mechanism and in Vero-E6 cells which has a defective type-1 IFN production, also suggesting a type-1 IFN independent mode of action. Molecular docking calculations demonstrated interactions of RO8191 with the CHIKV E proteins, corroborated by the ATR-FTIR assay, and with non-structural proteins, supported by the CHIKV-subgenomic replicon cells assay.

343 Endogenous double-stranded RNA is a potential target for psoriasis therapy

The activation of endogenous double-stranded RNA (endo-dsRNA) signalling pathways is linked to a variety of autoimmune diseases. Here, we investigated the role of endogenous dsRNA in psoriasis by quantifying dsRNA binding protein (DRBP) mRNA and mitochondrial dsRNA formation in response to narrow-band UVB (NB-UVB) treatment. RNAseq data from 34 psoriatic patients treated with NB-UVB were analysed. Biopsies were obtained from four sites 24h after NB-UVB irradiation (lesional unirradiated, nonlesional unirradiated lesional irradiated with one minimal erythemal dose (MED), lesional irradiated with three MEDs. Psoriatic lesional skin had a significantly higher expression of all examined DRBP (RIG, MDA5, LGP2, PKR, OAS1-3, OASL) as compared to nonlesional skin (increase between 1.5-21.7 fold, p <0.001 for all eight genes). NB-UVB treatment had no effect on the DRBPs after 24h of one or three MEDs of NB-UVB. Additionally, a significant reduction (about 20%) of dsRNA from mitochondrial transcripts was detected in response to both NB-UVB doses(p-value 0.012 and 0.001, respectively). These results indicate that increased levels of dsRNA activate RIG, MDA5, and LGP2 in lesional compared to nonlesional psoriasis skin. RIG, MDA5, and LGP2 in turn upregulate type1 IFN via recruiting TBK1, and IRF3/7, all of which were significantly upregulated in the lesional unirradiated skin. PKR binds endo-dsRNA, and activation of PKR enhances IFN-α/β expression via IRF3. Moreover, OASs trigger an antiviral response via a RIG induced IFN pathway. Once activated, IFN strongly enhances the transcription of DRBP thus creating a vicious cycle that exacerbates the immune response. In conclusion, upregulation of all DRBP genes in psoriatic lesional skin indicates that endo-dsRNA may play a crucial role in psoriasis pathogenesis, and the early reduction of the mitochondrial dsRNA after NB-UVB treatment suggests that dsRNA is an important target for psoriasis therapy.

Abstract 3512: Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity

Abstract Intrinsic malignant brain tumors, such as glioblastoma (GBM) are frequently resistant to immunotherapy with few hyper-mutated glioblastomas showing response. Here we have applied a long-term treatment with STING agonist and PD-1 inhibitors alone and in combination towards eliciting enhanced host immune response to combat GBM progression. To address these questions we generated mouse models of GBM by intracranial implantation of a murine GBM stem cell line or orthotopic CRISPR induced mutation to astrocytes. Systemic administration of STING agonist crosses the blood-brain barrier and induces a type-1 IFN signature in the brain and this was further enhanced in the tumor milieu. Treatment outcomes were addressed by MRi and treated animals showed a significant reduction in tumor volume and hence improved survival in comparison to the animals in the control group. Histochemical evaluation of tumor samples from the animals that received treatment showed elevated necrosis and aberrant/leaky vasculature in comparison to the control tumor samples. The presence of cytotoxic T-cells were similar between the groups but activated PD-1 positive T-cells were increased in treated tumors. Ex-vivo analysis of isolated T-cells from tumor baring mice confirms cytotoxic T-cells targeting the GBM cells. In conclusion, application of STING agonist promotes alteration of vasculitis and priming of T-cells in GBM results in increased survival and decreased tumor progression. These results reveal that GBM can be targeted by immune therapy through systemic activation of STING/IFN pathway. Citation Format: Justin V. Joseph, Huiqiang Cai, Brian Hansen, Soren R. Paludan, Mikkel Vendelbo, Martin K. Thomsen. Immunotherapy by STING activation in a CRISPR induce mouse model of GBM decrease tumor progression by CD8 T-cells and altered vascularity [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 3512.

Abstract CT240: LUMINOS-101: Phase 2 study of PVSRIPO with pembrolizumab in recurrent glioblastoma

Abstract Background: The prognosis for patients (pts) with recurrent (r) glioblastoma (GBM) is poor, with no highly effective approved therapies. Treatment failure may result from poor penetration of drugs through the blood-brain barrier and the immunosuppressive nature of the tumor microenvironment (TME). PVSRIPO, a recombinant poliovirus (PV):rhinovirus chimera, is a novel, non-neurovirulent, intratumoral immunotherapy. Trial results in rGBM pts showed greater long-term survival with PVSRIPO monotherapy (21%, 36-60 months [mos]) vs. criteria-matched external controls (4%, 36 mos; 2%, 60 mos; Desjardins 2018 NEJM). PVSRIPO targets CD155 (PV receptor), expressed on solid tumors and APC. PVSRIPO IT infection results in inflammatory-mediated destruction of tumor cells but non-lethal lingering infection in TME APC. This leads to a type-I/III interferon-dominant inflammation and ultimately, tumor antigen-specific T cell activation and recruitment (Brown 2017 Sci Transl Med), which is potentiated by immunologic recall to intratumoral replicating virus via prior vaccination. Induction of type-1 IFN dominant inflammation and compensatory activation of the PD-1:PD-L1 immune checkpoint (IC) pathway support investigation of PVSRIPO in combination with PD-1/L1 IC inhibitors. Immunologically cold mouse glioma models showed PVSRIPO+anti-PD-1 therapy resulted in greater anti-tumor response than either agent. Trial design/objectives: LUMINOS-101 (NCT04479241) is a phase 2, multicenter, open-label, single-arm study of intratumoral infusion of PVSRIPO (day 1: 5×107 TCID50) followed by the anti-PD-1 monoclonal antibody pembrolizumab (200mg IV q3w) in adult pts with rGBM. The trial objective is to evaluate the anti-tumor activity, safety, and tolerability of the combination. Eligibility criteria: Pts ≥18 years who had prior PV and boost IPOL® immunizations, histologically confirmed supratentorial rGBM, infusible 1 to ≤5.5cm enhancing disease, confirmed disease progression following prior therapies, and KPS ≥70 will be enrolled. Key exclusion criteria include multifocal disease; discontinuation of prior anti-PD-1/L1 agent for toxicity; prior intratumoral therapy, immunotherapy, or radiotherapy within 12 weeks; high-dose systemic corticosteroids; chemotherapy, anti-VEGF, or TTF therapy ≤1-6 weeks depending on the therapy; serious cerebral herniation syndrome; extensive leptomeningeal, subependymal, or ≥1cm enhancing disease crossing the midline; and severe active comorbidities. Endpoints: Primary endpoints are objective response rate, duration of response, and safety. Secondary endpoints include overall and progression-free survival and disease control rate and duration. Exploratory endpoints include assessment of tumor and blood for biomarkers of response. The study is currently enrolling in the US, and results will inform the design of a randomized phase 3 trial. Citation Format: Andrea True Kelly, Prakash Ambady, Michael Brown, Nicholas Butowski, Robert Cavaliere, William Curry, Annick Desjardins, Lisa Franklin, Henry Friedman, Matthias Gromeier, LeAnn Jackson, Lori Mixson, Shirley Ong, Dina Randazzo, Andrew Sloan, Patrick Wen, Garrett Nichols. LUMINOS-101: Phase 2 study of PVSRIPO with pembrolizumab in recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT240.

Abstract CT242: LUMINOS-103: A basket trial evaluating the safety and efficacy of PVSRIPO in patients with advanced solid tumors

Abstract Background: The recombinant poliovirus (PV):rhinovirus chimera, PVSRIPO, is a novel non-neurovirulent, intratumoral (IT) immunotherapy showing promising clinical activity in patients (pts) with recurrent glioblastoma and advanced anti-PD-1 refractory melanoma (Desjardins 2018 NEJM, Beasley 2020 SITC). PVSRIPO targets CD155 (PV receptor), expressed on most solid tumors (including bladder cancer [BC]) and antigen presenting cells (APCs) of the tumor microenvironment (TME). PVSRIPO infection results in inflammatory-mediated destruction of tumor cells but non-lethal lingering infection in TME APCs. This leads to a type-I/III interferon-dominant inflammation and ultimately, tumor antigen-specific T cell activation and recruitment (Brown 2017 Sci Transl Med), potentiated by immunologic recall to intratumoral replicating virus via prior vaccination. Induction of type-1 IFN dominant inflammation and compensatory activation of the PD-1:PD-L1 immune checkpoint (IC) pathway support investigation of PVSRIPO in combination with PD-1/L1 IC inhibitors. PVSRIPO+anti-PD-1 therapy resulted in greater anti-tumor response than either agent in immunologically cold mouse tumor models. Trial design, objectives, and eligibility criteria: LUMINOS-103 (NCT04690699) is a phase 1/2, open-label, multi-center, single-arm basket trial evaluating repeat administration of PVSRIPO (2×108 TCID50/mL)±anti-PD-1/L1 therapy in adults with solid tumors. Trial objectives are to evaluate the safety, tolerability, and anti-tumor activity of the combination in pts with solid tumors, beginning with muscle invasive bladder cancer (MIBC). The first arm will include pts in 2 cohorts: MIBC undergoing cystectomy (A); and unresectable locally advanced/metastatic BC (B). Safety/tolerability of PVSRIPO IT injections via cystoscopy will be evaluated in phase 1, Cohort A, and proceed to phase 2, where PVSRIPO+anti-PD-1/L1 therapy will be assessed in both cohorts. Eligible BC pts must refuse/be ineligible for cisplatin (A) or platinum-based therapy (B), have prior and boost PV immunization, and have measurable tumors (B) amenable to injection. Key exclusion criteria: other anti-cancer therapies ≤3 weeks, CNS metastases requiring active radiotherapy or steroids ≤14 days, systemic immunosuppressives ≤4 weeks, and severe active comorbidities. Endpoints: Primary endpoints include safety and tolerability and pathologic complete response (A) or overall response rate (B). Secondary endpoints: overall survival (both cohorts); for A, pathologic downstaging (p≤T2) and relapse-free survival; for B, duration of response and progression-free survival. Exploratory endpoints: assessment of tumor/blood biomarkers; measurement of disease by itRECIST. The trial will open US enrollment ≈ Q2 2021; results will inform later-stage trials in promising indications. Citation Format: Brant A. Inman, Arjun V. Balar, Matthew I. Milowsky, Raj S. Pruthi, Melissa J. Polasek, Shannon R. Morris, Lori Mixson, Kristin Orr, Elizabeth M. Woodson, Andrea True Kelly, Garrett Nichols. LUMINOS-103: A basket trial evaluating the safety and efficacy of PVSRIPO in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT242.

The analog of cGAMP, c-di-AMP, activates STING mediated cell death pathway in estrogen-receptor negative breast cancer cells.

Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negativebreast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.

Plasma checkpoint protein levels and galectin-9 in juvenile systemic lupus erythematosus.

SLE is a disease of the adaptive immune system where T lymphocyte dysfunction has an important role as well. We assessed the plasma levels of checkpoint receptors expressed on T cells, along with Galectin-9 to reflect type-1 IFN activity and IL-2Rα in childhood SLE patients. Forty-nine children with SLE and15 healthy controls were included. SLEDAI scores were evaluated at the time of sampling. CD25 (IL-2Rα), 4-1BB, B7.2 (CD86), TGF-β1, CTLA-4, PD-L1, PD-1, Tim-3, LAG- 3, Galectin-9 levels were studied by cytometric bead-based multiplex assay panel. Galectin-9 and PD-L1 were significantly higher in SLE patients. Other checkpoint proteins and IL-2Rα were also higher but did not reach statistical significance. There were significant correlations between SLEDAI and IL-2Rα, Galectin-9 and PDL1. There were three clinical clusters: Cluster 1 included patients with no major organ involvement, cluster 2 had predominantly haematological involvement(n=16) and cluster 3 (n=11) had predominantly renal involvement. Checkpoint proteins were not different among these three clusters. Our data supports that Galectin 9 and IL-2Rα are good markers for disease activity in childhood SLE. We need larger series to evaluate differences between disease clusters in SLE. We failed to show a significant correlation with checkpoint proteins and SLEDAI except for PDL1.

Rewiring of gene networks underlying mite allergen‐induced CD4 + Th‐cell responses during immunotherapy

Multiple regulatory mechanisms have been identified employing conventional hypothesis-driven approaches as contributing to allergen-specific immunotherapy outcomes, but understanding of how these integrate to maintain immunological homeostasis is incomplete. To explore the potential for unbiased systems-level gene co-expression network analysis to advance understanding of immunotherapy mechanisms. We profiled genome-wide allergen-induced Th-cell responses prospectively during 24months subcutaneous immunotherapy (SCIT) in 25 rhinitis, documenting changes in immunoinflammatory pathways and associated co-expression networks and their relationships to symptom scores out to 36months. Prior to immunotherapy, mite-induced Th-cell response networks involved multiple discrete co-expression modules including those related to Th2-, type1 IFN-, inflammation- and FOXP3/IL2-associated signalling. A signature comprising 109 genes correlated with symptom scores, and these mapped to cytokine signalling/T-cell activation-associated pathways, with upstream drivers including hallmark Th1/Th2- and inflammation-associated genes. Reanalysis after 3.5months SCIT updosing detected minimal changes to pathway/upstream regulator profiles despite 32.5% symptom reduction; however, network analysis revealed underlying merging of FOXP3/IL2-with inflammation-and Th2-associated modules. By 12months SCIT, symptoms had reduced by 41% without further significant changes to pathway/upstream regulator or network profiles. Continuing SCIT to 24months stabilized symptoms at 47% of baseline, accompanied by upregulation of the type1 IFN-associated network module and its merging into the Th2/FOXP3/IL2/inflammation module. Subcutaneous immunotherapy stimulates progressive integration of mite-induced Th cell-associated Th2-, FOXP3/IL2-, inflammation- and finally type1 IFN-signalling subnetworks, forming a single highly integrated co-expression network module, maximizing potential for stable homeostatic control of allergen-induced Th2 responses via cross-regulation. Th2-antagonistic type1 IFN signalling may play a key role in stabilizing clinical effects of SCIT.

Inverse correlation between serum complement component C1q levels and whole blood type-1 interferon signature in active tuberculosis and QuantiFERON-positive uveitis: implications for diagnosis.

ObjectivesTo examine the relation between serum C1q levels and blood type‐1 interferon signature (type‐1 IFN signature) in active pulmonary tuberculosis (APTB) and to determine whether combined measurement of serum C1q and type‐1 IFN signature may add to the diagnosis of QuantiFERON‐positive (QFT+) patients with uveitis of unknown cause.MethodsC1q was determined (ELISA) in serum from two distinct Indonesian cohorts, and in total, APTB (n = 72), QFT+ uveitis of unknown aetiology (n = 58), QFT− uveitis (n = 51) patients and healthy controls (HC; n = 73) were included. The type‐1 IFN signature scores were previously determined.ResultsSerum C1q was higher in APTB than HC (P < 0.001). APTB patients with uveitis had higher serum C1q than APTB patients without uveitis (P = 0.0207). Serum C1q correlated inversely with type‐1 IFN signature scores in APTB (P = 0.0036, r 2 = 0.3526), revealing that these biomarkers for active TB disease can be mutually exclusive. Stratification of QFT+ patients with uveitis of unknown cause, by serum C1q and type‐1 IFN signature, yielded four groups with different likelihood of suffering from active TB uveitis.ConclusionSerum C1q is elevated in APTB, especially in those cases with uveitis. We propose that combined measurement of blood type‐1 IFN signature and serum C1q may provide added value in the diagnosis of active TB disease. Combined measurement of type‐1 IFN signature and serum C1q in QFT+ patients without signs of active TB disease, but suffering from uveitis of unknown cause, may be of help to identify cases with low or high likelihood of having active TB uveitis, which may facilitate clinical management decisions.

Of Mice and Monkeys: Determining Protective Serological Titres in Model Zika Virus Infections

Developmental vaccines against emerging pathogens face many hurdles including determining what protective level of serological responses must be generated. Knowledge of a likely protective titre is critical where human challenge studies are not possible. We have used an anti-zika plasma pool from convalescent patients (candidate serological reference reagent NIBSC16/320-14), infused into cynomolgus macaques and Type-1 IFN deficient mice to determine likely protective neutralising titres. Anti-zika plasma was administered to a group of 4 macaques (single concentration) and groups of 8 A129 mice (4 group titration series) 24 hours prior to sub-cutaneous challenge with Zika virus PRVABC59. Plasma/sera samples were collected at regular intervals to track peripheral viremia and anti-zika antibody responses. FFPE tissues were collected at termination for histological analysis. Immediately prior to challenge, human IgG was detectable in all infused animals. Within macaques the NT50 at this time was 250. All macaques that received plasma were protected against zika virus infection as determined by plasma/tissue qRT-PCR and IgM responses. Titration within A129 mice gave a neutralisation titre of 110, above which mice were generally protected against zika infection. However this was not absolute as a small number of mice with high neutralisation levels were infected. A protective NT50 of 250 has been identified for macaques and this further titrated in A129 mice to give a guide protective neutralisation titre of 110. The lack of protection in some mice with higher titres is currently unclear and studies are underway to compare their infection pathology with that of controls.

Inflammatory cytokines responses of common carp, Cyprinus carpio, leucocytes in vitro treated by immunostimulants

Cytokines are important regulators of the immune system, and identifying fish cytokines has potential applications for the development of vaccines and/or immunostimulants application in fisheries. In order to understand the immune-related genes triggered by immunostimulants derived from pathogens, we investigated the effects of agonists (lipopolysaccharide (LPS), Poly I:C, and imiquimod) of three Toll-like receptor (TLR)—TLR4, TLR3, and TLR7, respectively—on the expression level of 10 cytokine genes—interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), CXC-chemokine, interleukin-10 (IL-10), type1 interferon (1INF), IFN-γ1, IFN-γ2, Mx protein, and interleukin-12 (IL-12p35 and IL-12 p40)—in the head kidney leukocytes of common carp (Cyprinus carpio L). All cytokine genes tested, except for type-1 IFN, were significantly up-regulated after LPS treatment. Poly I:C and imiquimod treatment resulted in striking up-regulation of most of the genes examined, particularly for the interferon genes as compared with the control groups. These results clearly demonstrate that TLRs agonists elicit the signaling pathways for cytokines production and initiation of innate immune responses in the common carp. The development of strategies to control the pathogen load and of immune-prophylactic measures must be addressed further to realize the economic potential of fish production. Key words: LPS, Poly I:C, Imiquimod, Carp, Cytokines.

Type1 IFN signaling on Tregs modulates the migration of Myeloid Derived Suppressor Cells

Abstract Multiple sclerosis is an autoimmune disorder whose severity is reduced with immune suppressive drugs. Therapeutic intervention with interferon-beta reduces disease exacerbations and delays relapses. The receptor for type 1 interferon, IFNAR, is present on virtually all cell types making it difficult to dissect the mechanisms involved. To address this issue, we induced experimental autoimmune encephalomyelitis (EAE) in mice with a conditional deletion (cKO) of IFNAR in T regulatory (Treg) cells (IFNARfl/flxFoxp3YFP-Cre). cKO mice developed severe EAE with an earlier onset than control mice. Although Treg cells from cKO mice appeared to be more activated, the activation status of and effector cytokine production by CD4+T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase, but higher in cKO CD4+T cells in the effector phase. Interestingly, we noted a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice, while generation of MDSCs in bone marrow and recruitment to spleen of WT and cKO mice were comparable. Surprisingly, we noted a decrease in the production of monocyte and granulocyte cell recruiting chemokine ligands by CD4+ and CD8+ T cells, B cells and endothelial cells in the dLN during the priming phase in the cKO mice. While modulation of Treg cell number and function by MDSCs has been documented, this study is one of the first to demonstrate that Treg cells modify the lymph node microenvironment by modulating MDSC homing and raises the possibility of a novel role for Treg cells in regulating the kinetics of MDSC recruitment during inflammatory conditions. This research was supported by the Intramural Research Program of NIAID, NIH.

The relationship between type 1 IFN and vasculopathy in anti-MDA5 antibody-positive dermatomyositis patients

Based on the antibody profiles of inflammatory myositis patients, we investigated the type 1 IFN (T1-IFN) signature in serum and DM skin to determine the relationship between T1-IFN and vasculopathy in anti-melanoma differentiation-associated 5 gene (MDA5) antibody-positive DM patients. We examined 47 patients with new-onset inflammatory myositis. We divided them into three groups: the anti-MDA5 antibody-positive patients (MDA5 group, n = 16), the anti-aminoacyl-tRNA synthetase antibody-positive patients (aminoacyl-tRNA synthetase group, n = 12), and the double-negative patients (n = 19). Serum T1-IFN signatures were revealed by a functional reporter assay, and we evaluated the T1-IFN signatures of skin based on Mx1 expression by immunohistochemistry. The numbers of patients with classical DM, clinically amyopathic DM and interstitial lung disease were 1, 15 and 13 in the MDA5 group, 2, 3 and 11 in the aminoacyl-tRNA synthetase group, and 10, 1 and 4 in the double-negative group, respectively. The signs of vasculopathies (i.e. palmer papules, skin ulcers and mononeuritis multiplex) were identified only in the MDA5 patients. Most of the MDA5 group showed the highest serum T1-IFN signatures among the three groups. In the histological analysis of DM skin, perivascular inflammations were significant in the MDA5 group. The MDA5 group's Mx1 expression was significantly strong, distributed in blood vessels and interstitial fibroblasts, and had spread to deep dermis. Anti-MDA5 antibody-positive DM patients showed high T1-IFN signatures in serum and affected skin. The high T1-IFN signatures of the MDA5 antibody-positive DM patients in serum and deep vasculatures suggested that T1-IFN may have important roles in the vasculopathy of these patients.

Abstract 5092: ZVex® lentiviral vector strongly activates pro-inflammatory, antigen processing, and anti-viral defense response pathways in monocyte-derived dendritic cells

Abstract Background: Dendritic cells (DCs) are professional antigen presenting cells that effectively bridge the innate and adaptive immune responses and require activation for successful priming of naïve T-cells. We have developed ZVex, an integration deficient, hybrid lentiviral vector engineered to target DC-SIGN expressed on immature myeloid DCs for genetic immunization against tumor antigens. As lentiviruses normally don't efficiently infect DCs, little is known about the functional effect of LV transduction of conventional DCs. Here, the effect of DC transduction with ZVex vectors was studied by gene expression profiling. Material and Methods: Human moDCs from multiple donors were prepared by 5-day stimulation with IL-4 and GM-CSF and transduced with ZVex-GFP or control vectors, such as a ZVex with nonfunctional reverse transcriptase (RT-dead), ZVex particles generated to contain no vector genome (empty ZVex), and a heat-inactivated ZVex preparation. Cellular RNA was isolated at different time points from transduced DC cultures and used for gene expression profiling with Nanostring’s human pan cancer immune panel (770 genes). Results: DCs transduced with ZVex vectors displayed statistically significant up-regulation of genes involved in pro-inflammatory, antigen processing, and anti-viral defense pathways. Noteworthy among the up-regulated genes were classical anti-viral response genes like OAS3, MX1, and interferon stimulated genes like ISG15 and ISG20. Incubation with the RT-dead mutant vector also led to up-regulation of these genes, albeit to a much lower extent, suggesting that LV-RNA itself can result in the activation of these pathways in DCs, though reverse transcription appears to further potentiate the response. Of note, cells transduced with empty ZVex, which consists of an intact virion particle but lacks encapsulated LV RNA, also mediated a low magnitude and breadth of DC transcriptional activation, possibly via signaling through DC-SIGN. Conclusions: ZVex incorporates several elements capable of inducing a potent innate immune activation in DCs. DCs are relatively insensitive to lentiviral infection, but the use of accessory proteins in ZVex makes DCs conducive to ZVex transduction and results in the induction of a Type-1 IFN response that seems to be largely dependent on reverse transcription. In addition, other engineered vector particle components such as non-reverse transcribed viral RNA, viral structural proteins, and/or engagement of the DC-SIGN receptor, also contribute to DC activation. Citation Format: Anshika Bajaj, Lisa Y. Ngo, Peter Berglund, Jan ter Meulen. ZVex® lentiviral vector strongly activates pro-inflammatory, antigen processing, and anti-viral defense response pathways in monocyte-derived dendritic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5092. doi:10.1158/1538-7445.AM2017-5092

Abstract 4563: Local and abscopal effects in oncolytic virotherapy are boosted by immune checkpoint blockade, immunogenic chemotherapy, or IFNAR blockade

Abstract Athough the clinical efficacy of oncolytic viruses has been demonstrated for local treatment, the ability to induce immune-mediated regression of distant metastases is still poorly documented. We here report that an engineered oncolytic Vaccinia Virus, VVWR-TK-RR--Fcu1, is able to induce an immunogenic cell death and thus to generate a systemic immune response. Effect on tumor growth and survival is largely driven by CD8+ T-cells, and we could demonstrate that the immune cell infiltrate in the tumor could be reprogrammed towards a higher ratio of effector T-cells to regulatory CD4+ T-cells. The key role of the type 1-IFN pathway in oncolytic virotherapy was also highlighted, and we could show a strong abscopal response in Ifnar-/- tumors. In this model, the single administration of the virus directly into the tumors, on one flank, led to regression in the contralateral flank (i.e. opposite to the virus injection site). Moreover, we observed that these effects are further enhanced when the oncolytic treatment is combined with either immunogenic chemotherapy such as oxaliplatin, or with immune checkpoint blockers such as anti-PD-1 or anti-CTLA-4. Altogether, these data suggest that local oncolytic virotherapy in patients with tumors altered in IFNAR signaling could increase immune-mediated abscopal regression of distant metastases. Citation Format: Laetitia Fend, Takahiro Yamazaki, Xavier Préville, Eric Quéméneur, Oliver Kepp, Julien Adam, Aurélien Marabelle, Jonathan Pitt, Guido Kroemer, Laurence Zitvogel. Local and abscopal effects in oncolytic virotherapy are boosted by immune checkpoint blockade, immunogenic chemotherapy, or IFNAR blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4563. doi:10.1158/1538-7445.AM2017-4563

Type I interferon signalling suppresses anti-parasitic CD4+ T cell responses during visceral leishmaniasis

Abstract Many pathogens, including viruses, bacteria, and protozoan parasites, suppress cell mediated immune responses through activation of type I Interferon (IFN-1) signalling. However the role of IFN-1 during Leishmania donovani infection causing visceral leishmaniasis (VL) is not well known. Here we report that IFN-1 play an important role in the pathogenesis of VL by impairing parasite clearance and suppressing pro-inflammatory cytokine production. Mice lacking type-1 IFN signalling (B6.IFNαR1−/− mice) had enhanced pro-inflammatory cytokine production and better control of parasite burden in liver as well as spleen compared to wild type C57BL/6 mice. IFN-1 signalling suppressed the CD4+ derived IFN-γ production and prevented Th1 response from controlling parasite replication. Experiment using bone marrow chimeric mice revealed that IFN-1 did not suppress CD4+ T via directly acting to this cell type. Using conventional dendritic cell (cDC) specific IFN-1 deficient mice (CD11c Cre-IFNARfl/fl), we observed that IFN-1 hampered CD4+ T cells response acting via dendritic cells. Studies in VL patients supported these findings and showed enhanced accumulation of mRNA encoding type I IFN signature genes in peripheral blood mononuclear cells (PBMCs) that were reduced following successful drug therapy. Critically, we also showed, using a whole blood assay, that blockade of type-1 IFN signalling enhanced antigen specific IFN-γ production, and that this response was HLA-II restricted. Together, these results identify the type-1 IFN signalling pathways as a potential therapeutic target to treat VL by stimulating anti-parasitic CD4+ T cell responses.

Imiquimod-induced CCR9 Ameliorates murine TNBS Colitis.

To investigate whether Imiquimod (IMQ) as TLR7 ligand protects mice from colonic inflammation and the mechanisms underlying in such immunoregulatory conditions. METHODS: Murine colitis was induced to Balb/c mice by administration of trinitrobenzene sulfonic acid (TNBS) with or without daily intraperitoneal administration of IMQ. Colitis was evaluated by body weight decreases and by histological score. Also colonic mRNA expression was measured by RT-PCR. To confirm the induction of Regulatory T cells (Tregs) by type-1 IFN from pDCs, we generated mouse bone marrow-derived pDCs and co-cultured these with CD4+ T cells isolated from mouse spleen with or without IMQ stimulation. Cytokine production in the culture supernatant was measured by ELISA and the number of Tregs were analyzed by flow cytometry. Spleen and mesenteric lymph nodes (MLN) from IMQ-treated mice were collected, and mRNA expressions of cytokine were measured by RT-PCR and cytokine productions were measured by ELISA. Tregs and chemokine expressions were analyzed in colon of TNBS-induced colitis mouse by immunohistochemistry. RESULTS: Administration of IMQ significantly suppressed colonic inflammation of TNBS-induced colitis. In the colons of IMQ-treated mice, mRNA expression of TNF-α was decreased, and strong expressions of IL-6, IFN-β and TGF-β were detected. IL-10 and TGF-β productions were increased in the supernatant of co-cultured cells stimulated with IMQ, although we were unable to detect Treg differentiaton in IMQ-stimulated co-cultured cells. In MLN of IMQ-treated mice, strong expressions of TLR7, IFN-β, TGF-β and Foxp3 mRNA were detected. IL-10 production from MLN cells was also increased in the IMQ-treated group. Finally, Tregs in the inflamed colon and CCR9 in MLN of IMQ-treated mice were detected. CONCLUSION: These results suggest that IMQ protects mice from TNBS colitis through induction of CCR9, which regulates accumulation of Tregs in the inflamed colon.

HIV-1-negative female sex workers sustain high cervical IFNɛ, low immune activation, and low expression of HIV-1-required host genes.

Sex workers within high HIV endemic areas are often a target population where anti-HIV prophylactic strategies are tested. We hypothesize that in women with high levels of genital exposure to semen changes in cervicovaginal mucosal and/or systemic immune activation will contribute to a decreased susceptibility to HIV-1 infection. To address this question, we assessed sexual activity, immune activation status (in peripheral blood), as well as cellular infiltrates and gene expression in ectocervical mucosa biopsies in female sex workers [FSW] (n=50), as compared to control women [CG] (n=32). FSW had low to absent HIV-1 specific immune responses with significantly lower CD38 expression on circulating CD4+ or CD8+ T-Cells (both: p<0.001) together with lower cervical gene expression of genes associated with leukocyte homing and chemotaxis. FSW also had increased levels of Interferon-ε gene and protein expression in the cervical epithelium together with reduced expression of genes associated with HIV-1 integration and replication. A correlative relationship between semen exposure and elevated type-1 IFN expression in FSW was also established. Overall, our data suggest that long-term condomless sex work can result in multiple changes within the cervicovaginal compartment that would contribute to sustaining a lower susceptibility for HIV-1 infection in absence of HIV-specific responses.