Presence Of IFN-α Research Articles

Inhibition of human cytomegalovirus replication by interferon alpha can involve multiple anti-viral factors.

The shortcomings of current direct-acting anti-viral therapy against human cytomegalovirus (HCMV) has led to interest in host-directed therapy. Here we re-examine the use of interferon proteins to inhibit HCMV replication utilizing both high and low passage strains of HCMV. Pre-treatment of cells with interferon alpha (IFNα) was required for robust and prolonged inhibition of both low and high passage HCMV strains, with no obvious toxicity, and was associated with an increased anti-viral state in HCMV-infected cells. Pre-treatment of cells with IFNα led to poor expression of HCMV immediate-early proteins from both high and low passage strains, which was associated with the presence of the anti-viral factor SUMO-PML. Inhibition of HCMV replication in the presence of IFNα involving ZAP proteins was HCMV strain-dependent, wherein a high passage HCMV strain was obviously restricted by ZAP and a low passage strain was not. This suggested that strain-specific combinations of anti-viral factors were involved in inhibition of HCMV replication in the presence of IFNα. Overall, this work further supports the development of strategies involving IFNα that may be useful to inhibit HCMV replication and highlights the complexity of the anti-viral response to HCMV in the presence of IFNα.

TYK2 Is Essential for IFNα-Induced Resolution of MPN Features in a Murine Jak2V617F PMF Model

Interferon (IFN)-α exhibits antiviral and antiproliferative effects on normal and neoplastic cells. It is an effective treatment option for myeloproliferative neoplasms (MPNs). The intracellular signaling of IFN-α is triggered by binding of IFN-α to its specific receptors on the cell surface, followed by the activation of tyrosine kinase-2 (TYK2) and Janus kinase-1 (JAK1), both of which are receptor-associated Janus kinases (JAKs). Activated JAKs activate many signaling molecules, including signal transducers and activators of transcription (STATs). Antiviral activity of IFN-α was abrogated in Stat1 deficient-embryonic fibroblasts, however, Tyk2 deficient-cells were resistant to viral infection in the presence of IFN-α, indicating the essential role of STAT1 for the antiviral activity of IFN-α, and TYK2 is not required for that. The antiproliferative effect is another major activity of IFN-α, however, the precise antiproliferative mechanism of IFN-α is not completely understood. We assessed the effect of ropeginterferon-α-2b, a monopegylated IFN-α-2b, on MPN model mice ( Jak2V617F mice), and investigated the role of TYK2 in the antiproliferative effect of IFN-α. Jak2VF mice exhibited leukocytosis and thrombocytosis, and TYK2 deficiency had no effect on MPN phenotypes induced by Jak2VF. IFN-α treatment was associated with a significant reduction in leukocyte and platelet counts in Jak2V617F mice. In contrast, leukocyte and platelet counts remained unchanged after IFN-α treatment in Jak2V617F and Tyk2 -/- mice. The proportions of BM progenitor cells as MEP and megakaryocyte lineage cells as MKP were markedly decreased by IFN-α treatment in Jak2V617F mice, whereas IFN-α had no effect on their proportions in Jak2V617F;T yk2 -/- mice. Next, we assessed the effects of IFN-a on cytokine-dependent colony formation. The colony numbers of CFU-GM and CFU-Meg from Jak2V617F mice decreased by about half in the presence of IFN-a. In contrast, cytokine-dependent colony formation was not affected by the presence of IFN-a in Jak2V617F; Tyk2 -/- mice. GSEA revealed significant enrichment of genes regulating antiproliferation in IFN-α-treated Jak2VF GMPs, but not in IFN-α-treated GMPs from Jak2V617F; Tyk2 -/- mice. Collectively, these results indicate that TYK2 plays an essential role in the antiproliferative effect of IFN-α on Jak2VF progenitors. IFN-α was reported to induce HSCs to enter the cell cycle. The significant reduction in quiescent cells and increase in cycling LT-HSCs in Jak2VF compared to WT HSCs after IFN-α treatment was reported (Mullally et al. Blood 2013). Consistent with previous reports, the proportion of long-term HSCs in BM from Jak2VF mice was decreased by 8-weeks-IFN-α treatment, however, this IFN-α effect was not observed when TYK2 was absent. In the pool of phenotypic long-term HSCs, there are subsets of stem cells with an intrinsic megakaryocytic bias and a propensity to commit directly to the megakaryocytic lineage. In the JAK2VF mutant fraction of HSCs from recipient mice transplanted with JAK2VF and WT BM cells, IFN-α treated mice were reported to show an increase in the percentage of CD41 high, megakaryocyte-skewed HSCs (Rao et al. Blood 2021). Consistent with this report, IFN-α treatment increased the proportion of CD41 highHSCs and decreased that of CD41 lowHSCs in BM from Jak2VF mice, and again, IFN-α-induced megakaryocytic bias of HSCs was not observed in TYK2 deficient HSCs. IFN-α had similar effect on Jak2VF-HSCs in recipient mice transplanted with Jak2VF and WT BM cells, and this IFN-α effect was not observed when TYK2 was absent. These observations indicate that TYK2 is essential for the effects of IFN-α on HSCs. These results indicate that TYK2 is indispensable for the effects of IFN-α on improvement of MPN features.

Monophosphoryl lipid A directly regulates Th1 cytokine production in human CD4+ T-cells through Toll-like receptor 2 and 4

BackgroundThe monophosphoryl lipid A (MPLA) is a detoxified LPS derivative and an emerging safe immune adjuvant in human vaccine development. The adjuvant MPLA promotes antigen-presenting cell (APC) function and preferentially induces a Th1 response following vaccination. However, the mechanism by which the MPLA detoxicates and exerts its adjuvants effect on T-cell, particualrly the Th1 response is unknown. AimsWe assessed the direct effects of MPLA on murine and human CD4+ T-cell proliferation and the profile of cytokine production ex vivo. ResultsWe report that CD4+ T-cells only express functional TLR2 and TLR4 when activated by TCR stimulation, in particularly in the presence of IFNα. The activated T cells thereafter can respond directly to MPLA. MPLA does not affect T-cell proliferation in human T cells, but can induce a balanced Th1 cytokine profile in CD4+ T-cells by reducing the production of Th1 cytokines and enhancing the production of the regulatory cytokine IL-10. The MPLA-mediated regulatory effect on activated CD4+ T-cells is TLR2 and TLR4 dependent and can be abolished by the lipid A blocker polymyxin B. ConclusionThese data provide evidence, at least in part, for the safe induction of an appropriate level of Th1 response by adjuvant MPLA in human vaccine development.

IFN-Alpha-Mediated Differentiation of Dendritic Cells for Cancer Immunotherapy: Advances and Perspectives.

The past decade has seen tremendous developments in novel cancer therapies through targeting immune-checkpoint molecules. However, since increasing the presentation of tumor antigens remains one of the major issues for eliciting a strong antitumor immune response, dendritic cells (DC) still hold a great potential for the development of cancer immunotherapy. A considerable body of evidence clearly demonstrates the importance of the interactions of type I IFN with the immune system for the generation of a durable antitumor response through its effects on DC. Actually, highly active DC can be rapidly generated from blood monocytes in vitro in the presence of IFN-α (IFN-DC), suitable for therapeutic vaccination of cancer patients. Here we review how type I IFN can promote the ex vivo differentiation of human DC and orientate DC functions towards the priming and expansion of protective antitumor immune responses. New epigenetic elements of control on activation of the type I IFN signal will be highlighted. We also review a few clinical trials exploiting IFN-DC in cancer vaccination and discuss how IFN-DC could be exploited for the design of effective strategies of cancer immunotherapy as a monotherapy or in combination with immune-checkpoint inhibitors or immunomodulatory drugs.

Abstract 3233: Reduced inflammatory cytokine production by CAR-modified Th1/Tc1-polarized T-Rapa cells is not sufficient to reduce monocyte-driven production of IL-6

Abstract Introduction: Chimeric antigen receptor (CAR) engineered T cells have delivered remarkable anti-tumor effects. However, the potential for severe and life-threatening cytokine release syndrome (CRS) represents a therapeutic obstacle. CRS is in large part IL-6 mediated, as indicated by the recent FDA approval of tocilizumab after CAR-T therapy. However, because CRS remains a significant clinical toxicity, additional approaches are necessary. Previous experimental data has shown that T-Rapa cells (T cells grown ex vivo in rapamycin) secrete reduced levels of some cytokines, and express a central memory phenotype that is associated with increased in vivo persistence. In the clinical context, allogeneic T-Rapa cells of a Th2 phenotype were associated with a low rate of GVHD, which is, in part, a cytokine-mediated syndrome. As such, T-Rapa cells represent a novel CAR effector cell type (CAR-T-Rapa). We hypothesized that Th1/Tc1 polarized CAR-T-Rapa cells would mediate efficient cytotoxicity, secrete reduced inflammatory T cell cytokines, and thereby result in a reduced propensity for IL-6 secretion, which is predominantly a monocyte-derived cytokine. Methods: Human CD3+ cells were treated with rapamycin in the presence of IFN-α and IL-2 to produce T-Rapa cells with a Th1/Tc1 phenotype, and were transduced with a CD19-41BB-CD3ζ CAR. T cells were expanded and used in assays including FACS assessment of T cell phenotype, co-culture assays, and 51Cr release assays, in comparison with non-rapamycin treated CAR T cells and non-transduced controls. ELISA and Luminex were used for assessment of cytokine levels in cell culture supernatants. Results: CAR-T and CAR-T-Rapa cells exhibited equal levels of cytotoxicity against CD19+ tumor cell lines. Both CAR-T and CAR-T-Rapa cells produced comparable amounts of IL-2 and undetectable IL-6 following co-culture with CD19+ tumor cell lines. However, CAR-T-Rapa cells produced significantly less IFN- γ, IL-4, TNF-α, and GM-CSF than CAR-T cells. When T cells were co-cultured with both tumor cells and monocytes, a high level of IL-6 secretion was detected in both CAR-T and CAR-T-Rapa conditions. Conclusions: CAR-T-Rapa cells are effective killers, while producing considerably less inflammatory cytokines in comparison to CAR-T cells. However, in the monocyte-replete condition, CAR-T-Rapa cells did not result in reduced IL-6 secretion, thereby indicating that controlling T cell inflammatory cytokine levels may not be sufficient to mitigate CRS. Citation Format: Robyn A. Oldham, Tania Felizardo, Nathaniel Zhu, Daniel H. Fowler, Jeffrey A. Medin. Reduced inflammatory cytokine production by CAR-modified Th1/Tc1-polarized T-Rapa cells is not sufficient to reduce monocyte-driven production of IL-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3233.

Impact and prognosis of the expression of IFN-α among tuberculosis patients.

Mycobacterium tuberculosis (M.tb) infection stimulates the release of cytokines, including interferons (IFNs). IFNs are initiators, regulators, and effectors of innate and adaptive immunity. Accordingly, the expression levels of Type I (α, β) and II (γ) IFNs, among untreated tuberculosis (TB) patients and household contacts (HHC) clinically free of TB was assessed. A total of 264 individuals (TB patients-123; HHC-86; laboratory volunteers-55; Treated TB patients-36) were enrolled for this study. IFN-α mRNA expression levels predominated compared to IFN-γ and IFN-β among untreated TB patients. IFN-α transcripts were ~3.5 folds higher in TB patients compared to HHC, (p<0.0001). High expression of IFN-α was seen among 46% (56/ 123) of the TB patients and 26%, (22/86) of HHCs. The expression levels of IFN-α correlated with that of IFN transcriptional release factor 7 (IRF) (p<0.0001). In contrast, an inverse relationship exists between PGE2 and IFN-α expression levels; high IFN-α expressers were associated with low levels of PGE2 and vice-versa (Spearman's rho = -0.563; p<0.0001). In-vitro, IFN-α failed to restrict the replication of intracellular M.tb. The anti-mycobacterial activity of IFN-γ was compromised in the presence of IFN-α, but not by IFN-β. The expression of IFN-α and β diminished or is absent, among successfully treated TB patients. These observations suggest the utility of assessment of Type I IFNs expression levels as a prognostic marker to monitor tuberculosis patient response to chemotherapy because changes in Type I IFNs expression are expected to precede the clearance and /reduction in bacterial load.

INFLUENCE OF DEXAMETHASONE-MODIFIED DENDRITIC CELLS GENERATED WITH IFNα UPON AUTOLOGOUS T LYMPHOCYTE FUNCTIONS IN THE PATIENTS WITH RHEUMATOID ARTHRITIS

Dendritic cells (DCs) play a key role in maintaining the peripheral tolerance of lymphocytes to autoantigens. Recovery of immunological tolerance in autoimmune diseases, particularly, in rheumatoid arthritis (RA) is considered a new therapeutic strategy. The aim of this work was to study the effect of dexamethasone-modified DCs generated from monocytes of RA patients in the presence of IFNα (DCsDex), upon autologous T lymphocytes in mixed leukocyte culture (auto-MLC), and to investigate possible mechanisms of the DCsdextolerogenic effect upon autoreactive T cells. We have shown, that DCsDexfrom RA patients induce T cell hyporeactivity in auto-MLC. Hyporeactivity of T cells is associated with cell cycle blockage in CD4+T lymphocytes and decreased IFNγ, IL-17, IL-4 and IL-13 production, which indicates the induction of CD4+T cell anergy. In this case, inhibition of Th1/Th17 has been more pronounced than the suppression of Th2 cells producing IL-4 and IL-13. Along with T cell anergy, the decrease of proliferative response in auto-MLC is associated with increased CD3+T lymphocyte apoptosis. In addition, the DCsDexof RA patients suppresses the proliferation of autologous T cells stimulated by unmodified DCs. This effect is associated with enhancement of IL-10-producing CD4+T cells in the auto-MLC, thus being indicative for an ability of DCsDexto induce conversion of CD4+T lymphocytes into regulatory T cells (Tr1). The data obtained characterize a new type of tolerogenic DCs, generated from blood monocytes of RA patients in the presence of IFNα and modified by dexamethasone, thus revealing a mechanism for tolerogenic effect of DCsDexupon T cells that recognize self-antigens in auto-MLC.

The Ubiquitin-Specific Protease 18 Promotes Hepatitis C Virus Production by Increasing Viral Infectivity.

Background and Aims Ubiquitin-specific protease 18 (USP18) is involved in immunoregulation and response to interferon- (IFN-) based treatment in patients chronically infected with hepatitis C virus (HCV). We investigated whether and how its upregulation alters HCV infection. Methods Overexpression of wild-type (USP18 WT) or catalytically inactive mutant (USP18 C64S) USP18 was examined for effects on HCV replication in the absence and presence of IFNα or IFNλ using both the HCV-infective model and replicon cells. The IFN signaling pathway was assessed via STAT1 phosphorylation (western blot) and downstream ISG expression (real-time PCR). Mechanistic roles were sought by quantifying microRNA-122 levels and J6/JFH1 infectivity of Huh7.5 cells. Results We found that overexpression of either USP18 WT or USP18 C64S stimulated HCV production and blunted the anti-HCV effect of IFNα and IFNλ in the infective model but not in the replicon system. Overexpressed USP18 showed no effect on Jak/STAT signaling nor on microRNA-122 expression. However, USP18 upregulation markedly increased J6/JFH1 infectivity and promoted the expression of the key HCV entry factor CD81 on Huh7.5 cells. Conclusions USP18 stimulates HCV production and blunts the effect of both type I and III IFNs by fostering a cellular environment characterized by upregulation of CD81, promoting virus entry and infectivity.

The Transcriptional and Protein Profile From Human Infected Neuroprogenitor Cells Is Strongly Correlated to Zika Virus Microcephaly Cytokines Phenotype Evidencing a Persistent Inflammation in the CNS.

Zika virus (ZIKV) infection during pregnancy is associated with microcephaly, a congenital malformation resulting from neuroinflammation and direct effects of virus replication on the developing central nervous system (CNS). However, the exact changes in the affected CNS remain unknown. Here, we show by transcriptome analysis (at 48 h post-infection) and multiplex immune profiling that human induced-neuroprogenitor stem cells (hiNPCs) respond to ZIKV infection with a strong induction of type-I interferons (IFNs) and several type-I IFNs stimulated genes (ISGs), notably cytokines and the pro-apoptotic chemokines CXCL9 and CXCL10. By comparing the inflammatory profile induced by a ZIKV Brazilian strain with an ancestral strain isolated from Cambodia in 2010, we observed that the response magnitude differs among them. Compared to ZIKV/Cambodia, the experimental infection of hiNPCs with ZIKV/Brazil resulted in a diminished induction of ISGs and lower induction of several cytokines (IFN-α, IL-1α/β, IL-6, IL-8, and IL-15), consequently favoring virus replication. From ZIKV-confirmed infant microcephaly cases, we detected a similar profile characterized by the presence of IFN-α, CXCL10, and CXCL9 in cerebrospinal fluid (CSF) samples collected after birth, evidencing a sustained CNS inflammation. Altogether, our data suggest that the CNS may be directly affected due to an unbalanced and chronic local inflammatory response, elicited by ZIKV infection, which contributes to damage to the fetal brain.

CAR-Modified Th1/Tc1-Polarized T-Rapa Cells Dissociate Inflammatory Cytokine Secretion from Anti-Tumor Cytotoxicity

▪Introduction: Despite some striking clinical success thus far, chimeric antigen receptor (CAR) engineered cells have the potential to cause severe side effects. Neurotoxicity and cytokine release syndrome (CRS), the latter characterized by increased levels of cytokines such as IL-6, IFN-γ, and MCP-1, are common adverse events associated with CAR therapy. Lymphodepleting preconditioning regimens are associated with improved clinical responses to CAR therapy, yet lymphodepletion has also been identified as a risk factor for CRS. Understanding and management of these toxicities has improved significantly, however these conditions are challenging to treat and can be life-threatening. The ability to limit or prevent initiation of CRS would greatly improve the safety of CAR therapy.Previous clinical trials have shown that T-Rapa cells (patient T cells that have been grown exvivo in rapamycin) can be successfully infused back into autologous recipients after a low-dose conditioning regimen. After infusion, these T-Rapa cells have potent effector functions and demonstrate long-term persistence. Here we determine that T-Rapa cells, engineered by lentivirus-mediated gene transfer to express an anti-CD19 CAR, are just as effective at killing tumor cells as similarly-engineered pan T cells but produce dramatically less IFN-γ, for example.Methods: Human CD3+ cells were treated with rapamycin in the presence of IFN-α and IL-2 to produce T-Rapa cells with a Th1/Tc1 phenotype. An anti-CD19-41BB-CD3ζ CAR construct was subcloned into a lentiviral vector backbone containing an IRES-eGFP element. Vector was prepared and used to transduce T or T-Rapa cells. Transgene expression was assessed by FACS for eGFP and Protein L staining for the CAR. CAR-T cells were then expanded using CD3/CD28 beads in the presence of IL-2. The expanded cells were used in assays including FACS assessment of T cell phenotype, co-culture assays, and 51Cr release assays in comparison with non-rapamycin treated CAR T cells and non-transduced controls.Results: Following transduction and expansion, similar eGFP and CAR expression levels were found in T and T-Rapa cells transduced at the same MOI. CAR-T and CAR-T-Rapa cells developed from multiple independent T cell donors exhibited similar phenotypes at days 5 and 14 post-thaw, as determined by analyses of T-cell subset and exhaustion markers including CD45RO, CD127, CCR7, CD95, CD25, CXCR3, CTLA-4, PD-1, LAG-3, and TIM-3. Both CAR-T and CAR-T-Rapa cells exhibited comparable levels of cytotoxicity against CD19+ Raji, SUP-B15 and RS4;11 cancer cell lines after coculture for 4 hours in a 51Cr release assay. Further, both T and T-Rapa CAR cells produced similar amounts of IL-2 following a 24-hour coculture with CD19+ Raji, SUP-B15 and RS4;11 cancer cell lines, as measured by ELISA. Interestingly, CAR-T-Rapa cells produced significantly less IFN- γ that CAR-T cells after 24 hours of coculture with CD19+ tumor cells. This observation was consistent for CAR-T and CAR-T-Rapa cells assessed at both day 5 and day 14 post-thaw.Conclusions: T-Rapa cells can be successfully transduced with a CAR vector, and show comparable T cell subset, exhaustion phenotype, and cytotoxicity to CAR-T cells that have not been treated with rapamycin. In spite of these similarities, when challenged with CD19+ tumor cells, CAR-T-Rapa cells produced less IFN-γ than CAR-T cells. Decreased production of IFN- γ may reduce the risk and severity of CRS, improving the safety of CAR therapy. Additional cytokine production studies, as well as in vivo studies, are underway to further characterize T-Rapa cells as a novel CAR effector cell type. DisclosuresFelizardo:Rapa Therapeutics: Employment, Patents & Royalties. Zhu:Rapa Therapeutics: Employment, Patents & Royalties. Fowler:Rapa Therapeutics: Employment, Equity Ownership, Patents & Royalties. Medin:Rapa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Class 1 HDAC and HDAC6 inhibition inversely regulates CD38 induction in myeloma cells via interferon-α and ATRA.

Class 1 HDAC and HDAC6 inhibition inversely regulates CD38 induction in myeloma cells via interferon-α and ATRA.

Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection.

T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR−/−) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections.

Modulation of type I interferon signaling by African swine fever virus (ASFV) of different virulence L60 and NHV in macrophage host cells

ASFV causes an important disease of domestic swine and wild boar. Currently no vaccine is available, highlighting the necessity to understand ASFV modulation of innate immune responses in natural host cells. With this aim, macrophage cultures enriched in SWC9 and CD163 differentiation markers were infected in parallel with high virulent ASFV/L60 and low virulent ASFV/NHV, the latter lacking MGF 360 and 505/530 genes associated with type I interferon (IFN I) control. IFN I production and signaling were studied after completion of the viral cycles. None of the viruses increased IFN I production in host cells, and accordingly, didn't cause activation of the central mediator of the pathway IRF3. However, upon stimulation by poly:IC treatment during infections, L60 and NHV similarly inhibited IFN I production. This didn't seem to depend on IRF3 modulation since its activation levels were not significantly decreased in L60 infection and were even increased in NHV’s, in comparison to stimulated mock infections. The infections didn't evidently activate JAK-STAT pathway mediators STAT1 and STAT2, but did increase expression of interferon stimulated genes (ISGs), to higher levels in NHV than L60 infection. Interestingly, in presence of IFN-α, L60 but not NHV was able to decrease significantly the expression of some of the ISGs tested. Overall, both L60 and NHV were able to inhibit IFN I production in macrophages, through a mechanism not dependent on IRF3 modulation. The high virulent isolate showed however a more effective control of the downstream ISGs expression pathway.

Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine

Type I interferons, such as interferon alpha (IFN-α), contribute to innate antiviral immunity by promoting production of antiviral mediators and are also involved in promoting an adaptive immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating and costly viruses to the swine industry world-wide and has been shown to induce a meager IFN-α response. Previously we administered porcine IFN-α using a replication-defective adenovirus vector (Ad5-IFN-α) at the time of challenge with virulent PRRSV and demonstrated an increase in the number of virus-specific IFNγ secreting cells, indicating that the presence of IFN-α at the time of infection can alter the adaptive immune responses to PRRSV. In the current experiment, we explored the use of IFN-α as an adjuvant administered with live-attenuated PRRSV vaccine as a method to enhance immune response to the vaccine. Unlike the previous studies with fully virulent virus, one injection of the Ad5-IFN-α abolished replication of the vaccine virus and as a result there was no detectible adaptive immune response. Although IFN-α did not have the desired adjuvant effect, the results further highlight the use of IFN-α as a treatment for PRRSV infection.

Abstract 951: Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity

Abstract Background: Infiltration of cytotoxic T-cells (CTL) in tumors is known to be associated with improved patient’s clinical outcomes. In contrast, local infiltration with regulatory T-cells (Treg) predicts accelerated progression and shorter overall survival. The above observations highlight the need for new measures to promote selective accumulation of CTLs but not Tregs in tumor microenvironments (TME). Here we compare the TME-modulating impact of poly-I:C with a selective TLR3 ligand, rintatolimod showing an induction of CCL22, CXCL12 and expression of COX-2 and downstream suppressive factors, selectively by poly-I:C. Finally, we report on the optimized combinatorial adjuvant to reprogram tumor microenvironment. Materials and Methods: Twelve ovarian cancer specimens were cultured in the presence of IFNα, indometacin (COX-1/2 inhibitor) or/and one of two synthetic TLR3 ligands, poly-I:C (non-selective activator of TLR3 and helicases) or rintatolimod (selective TLR3 ligand). Biopsies were harvested for mRNA measurements and culture supernatants were analyzed for CCL5, CXCL10 and CCL22 concentrations. Alternatively, established ovarian cancer cell lines, monocyte-derived macrophages, THP1 cells or adult fibroblasts were used in analogous experiments. Chemotaxis assays were performed using pre-activated CD8+ T-cells (top chamber) and supernatants from the differentially treated ovarian cancer specimens. Western blot experiments were used to evaluate the impact of NFκB. Blocking experiments using soluble TNFα receptor inhibitor were performed. Results: Both, Poly-I:C and rintatolimod, induced CTL attractant CXCL10. Unexpectedly, poly-I:C but not rintatolimod promoted the expression of the MDSC/Treg attractant CXCL12, which was reversed by addition of indometacin. Furthermore, poly-I:C induced expression of COX-2 and COX-2-dependent suppressive factors (IDO, IL-10). Evaluating the COX-2 cascade, TNFα was found to be increased in the presence of poly-I:C but not rintatolimod. Additionally, Western Blot analysis showed I-κB degradation (as a marker of NF-κB activation), selectively in response to poly-I:C. We observed that the combination of both TLR-3 ligands with IFNα and indomethacin selectively induced the desirable chemokines CCL5 and CXCL10 and suppressed CCL22 in tumor samples with similar results in the macrophages and fibroblasts. The ovarian cancer cell lines showed only minimal expression of chemokines. Conclusion: We demonstrate the feasibility of selective modulation of TME, using different clinically applicable factors and their combinations. We show for the first time, that poly-I:C, but not a selective TLR3 ligand, induces NFκB and undesirable COX2-dependent suppressive factors, which though can be eliminated through addition of a COX-1/2 inhibitor. These effects may be avoided using a selective TLR3 ligand. Citation Format: Marie-Nicole Theodoraki, Ravi Muthuswamy, Jamie Voyten, Francesmary Modugno, Natasa Obermajer, Robert P. Edwards, Pawel Kalinski. Induction of COX-2 and NFκB activation represent TLR3-independent undesirable aspects of poly-I:C activity [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 951. doi:10.1158/1538-7445.AM2017-951

INDUCTION OF T-CELL IMMUNE RESPONSE IN CHRONIC HCV-INFECTED PATIENTS WITH UNDERLYING DENDRITIC CELL IMMUNOTHERAPY

A key role of T cells in viral elimination and absence of strong T cell responses in patients with chronic hepatitis C virus (HCV) infection presumes that activation of antigen-specific T cells may be a promising approach to enhance treatment efficacy. Given the central role of dendritic cells (DCs) in the induction of T cell response, the aim of our study was to evaluate effects of DC immunotherapy upon immunological parameters in chronic HCV infection. Ten patients with chronic hepatitis C (genotype 1) were vaccinated with monocytederived DCs, generated in presence of IFNα (IFN-DCs) and pulsed with recombinant HCV Core (1–120) and NS3 (1192–1457) proteins. The vaccination protocol included as initiating procedure (one injection per week, ns = 4) and maintaining treatment (one monthly injection, ns = 6), with subsequent follow up for 6 months. The immunotherapy was not associated with serious adverse events, significant post-vaccination reactions, or increased hepatitis C activity, according to biochemical tests. Ex vivo studies have shown that immunotherapy elicited strong and stable immune response to Core and moderate response to NS3 protein, which manifested as a significant increase of MNC proliferation and IFNγ production in response to Core and enhancement of IFNγ production (without higher proliferation rates), in response to NS3. DC immunotherapy also led to increase of ConA-induced MNC proliferation up to normal levels indicating a recovery of mitogenic T cell reactivity. Meanwhile, T cell activation did not elicit antigen-specific Th2 response and expansion of CD4 + CD25 + CD127 regulatory T cells. Despite induced immune response, the immunotherapy with DCs was not accompanied by decreased viremia levels. Nevertheless, a transitory decrease of viral load in four patients and stable decrease of viremia in two patients as well as an inverse correlation between NS3-specific proliferation and viremia (Rss = 0.62; p < 0.05) by the end of 6-month follow-up indicated that the antigenspecific T cells may have a potential to control viral replication.

Oncolysate-loaded Escherichia coli bacterial ghosts enhance the stimulatory capacity of human dendritic cells.

The natural adjuvant properties of bacterial ghosts (BGs) lie within the presence of intact pathogen-associated molecular patterns on their surface. BGs can improve the direct delivery, natural processing and presentation of target antigens within dendritic cells (DCs). Moreover, sensitization of human DCs by cancer cell lysate (oncolysate)-loaded BGs in the presence of IFN-α and GM-CSF enhanced DC maturation as indicated by an increased expression of maturation markers and co-stimulatory molecules, higher production of IL-12p70 and stimulation of significantly increased proliferation of both autologous CD4+ and CD8+ T cells compared to DCs matured in the presence of purified lipopolysaccharide. The induced T cells efficiently recognized oncolysate-derived tumor-associated antigens expressed by cancer cells used for the production of oncolysate. Our optimized one-step simultaneous antigen delivery and DC maturation-inducing method emerges as a promising tool for the development and implementation of next-generation cellular cancer immunotherapies.

Genetic and phenotypic analyses of sequential vpu alleles from HIV-infected IFN-treated patients

Treatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties.

IDO1 and TGF-β Mediate Protective Effects of IFN-α in Antigen-Induced Arthritis.

IFN-α prevents Ag-induced arthritis (AIA), and in this study we investigated the role of IDO1 and TGF-β signaling for this anti-inflammatory property of IFN-α. Arthritis was induced by methylated BSA (mBSA) in mBSA-sensitized wild-type (WT), Ido1-/-, or Ifnar-/- mice, treated or not with IFN-α or the IDO1 product kynurenine (Kyn). Enzymatic IDO1 activity, TGF-β, and plasmacytoid dendritic cells (pDC) were neutralized by 1-methyltryptophan and Abs against TGF-β and pDC, respectively. IDO1 expression was determined by RT-PCR, Western blot, and FACS, and enzymatic activity by HPLC. Proliferation was measured by 3H-thymidine incorporation and TGF-β by RT-PCR and ELISA. WT but not Ido1-/- mice were protected from AIA by IFN-α, and Kyn, the main IDO1 product, also prevented AIA, both in WT and Ifnar-/- mice. Protective treatment with IFN-α increased the expression of IDO1 in pDC during AIA, and Ab-mediated depletion of pDC, either during mBSA sensitization or after triggering of arthritis, completely abrogated the protective effect of IFN-α. IFN-α treatment also increased the enzymatic IDO1 activity (Kyn/tryptophan ratio), which in turn activated production of TGF-β. Neutralization of enzymatic IDO1 activity or TGF-β signaling blocked the protective effect of IFN-α against AIA, but only during sensitization and not after triggering of arthritis. Likewise, inhibition of the IDO1 enzymatic activity in the sensitization phase, but not after triggering of arthritis, subdued the IFN-α-induced inhibition of mBSA-induced proliferation. In conclusion, presence of IFN-α at Ag sensitization activates an IDO1/TGF-β-dependent anti-inflammatory program that upon antigenic rechallenge prevents inflammation via pDC.

NK Cell Activation in the Antitumor Response Induced by IFN-α Dendritic Cells Loaded with Apoptotic Cells from Follicular Lymphoma Patients.

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma. This malignancy is considered virtually incurable, with high response rates to therapy but frequent relapses. We investigated the ability of monocyte-derived dendritic cells generated in the presence of IFN-α and GM-CSF (IFN-DC) and loaded with apoptotic lymphoma cells to activate immune responses against FL cells, with the ultimate goal of designing novel patient-specific vaccination strategies for the treatment of FL. In this article, we show that apoptotic tumor cell-loaded IFN-DC from FL patients, which were cultured for 2 wk with autologous lymphocytes, led to Th1 response skewing, based on significantly higher levels of IFN-γ production and a remarkable increase in CD8(+) and NK cell frequency, consistent with the detection of enhanced cytotoxic effector function toward autologous FL cells. IFN-DC were found to promote efficient NK cell activation, increased expression of cytotoxicity receptors, and extensive IFN-γ production in the virtual absence of IL-10. Moreover, direct recognition and killing of primary autologous lymphoma cells by activated NK cells from FL patients was also demonstrated. A critical role was demonstrated for MHC class I-related chain A and B and membrane-bound IL-15 in IFN-DC-mediated NK cell activation and early IFN-γ production. The overall results indicate that IFN-DC loaded with autologous apoptotic FL cells represent a valuable tool for improving the potency of therapeutic cancer vaccines through the efficient induction of NK cell activation and promotion of CD8(+) T cell antitumor immunity.