Blockade Of IFN-γ Research Articles

Lentinan inhibits tumor angiogenesis via interferon \u03b3 and in a T cell independent manner

BackgroundAntiangiogenic agents are commonly used in lung and colon cancer treatments, however, rapid development of drug resistance limits their efficacy.MethodsLentinan (LNT) is a biologically active compound extracted from Lentinus edodes. The effects of LNT on tumor angiogenesis were evaluated by immunohistochemistry in murine LAP0297 lung and CT26 colorectal tumor models. The impacts of LNT on immune cells and gene expression in tumor tissues were determined by flow cytometry, qPCR, and ELISA. Nude mice and IFNγ blockade were used to investigate the mechanism of LNT affecting on tumor angiogenesis. The data sets were compared using two-tailed student’s t tests or ANOVA.ResultsWe found that LNT inhibited tumor angiogenesis and the growth of lung and colon cancers. LNT treatments elevated the expression of angiostatic factors such as IFNγ and also increased tumor infiltration of IFNγ-expressing T cells and myeloid cells. Interestingly, IFNγ blockade, but not T cell deficiency, reversed the effects of LNT treatments on tumor blood vessels. Moreover, long-lasting LNT administration persistently suppressed tumor angiogenesis and inhibited tumor growth.ConclusionsLNT inhibits tumor angiogenesis by increasing IFNγ production and in a T cell-independent manner. Our findings suggest that LNT could be developed as a new antiangiogenic agent for long-term treatment of lung and colon cancers.

Systemic juvenile idiopathic arthritis and macrophage activation syndrome: update on pathogenesis and treatment.

The past decade has seen substantial progress in defining the cause and pathogenesis of the chronic childhood arthropathy systemic juvenile idiopathic arthritis (SJIA) and its related complication macrophage activation syndrome (MAS). The purpose of this review is to describe and synthesize advances in this field, particularly since 2016, with the potential to transform clinical practice. Newly developed MAS classification criteria have been further studied and validated in other diseases and populations, as well as a recently proposed score to distinguish MAS from familial hemophagocytic lymphohistiocytosis. There has also been substantial progress toward understanding the genetic underpinnings of SJIA and MAS, both through targeted study of specific genes and the results of a large genome-wide association study. The immunopathogenesis of SJIA has been further elucidated through several studies regarding the proinflammatory cytokines interleukin-18, interferon (IFN)γ, and how their interplay impacts emergence of MAS. Finally, big data studies integrating genomic information with immunophenotypes have potential to provide novel insights into disease mechanisms in SJIA. Collectively, these research advances have significant implications regarding the classification and diagnosis of SJIA and MAS, and support a next generation of biologic treatments including kinase inhibitors and targeted interleukin-18 or IFNγ blockade.

Combined neutralization of interferon gamma and tumor necrosis factor alpha induces IL-4 production but has no direct additive impact on parasite burden in splenic cultures of human visceral leishmaniasis.

Immune activating cytokines Interferon (IFN)-γ and Tumor necrosis factor (TNF)-α are known to activate macrophages for killing of Leishmania parasite. IFN-γ provides therapeutic potential while TNF-α has been recognized to mediate protection in visceral model of infection. In the present study we investigated whether combination of IFN-γ and TNF-α has better therapeutic strength than individually using one of these cytokines in Visceral Leishmaniasis (VL) patients. We performed combined blockade of IFN-γ and TNF-α in VL splenic biopsies and demonstrated it’s impact on number of viable amastigotes and cytokine production. Additionally, selective depletion of splenic cell subsets was performed to establish the cellular sources of IFN-γ and TNF-α. Treatment of splenic aspirate cells with combination of anti-IFN-γ and anti-TNF-α monoclonal antibodies for 72 hours enabled no direct additive impact of these cytokines on parasite replication and IL-10 secretion, but IL-4 production was induced. Further assessment of splenic biopsies put forward CD4+ T cells as a source of IFN-γ whereas CD14+ cells contribute towards TNF-α production. Overall our results suggest, the interplay of pro-inflammatory cytokines IFN-γ derived from CD4+T lymphocytes and TNF-α from CD14+ cells has no direct additive impact on parasite replication but induces IL-4 production. Our data does not support direct targeting of IFN-γ and TNF-α for combination therapy but targeting these cytokines as an adjuvant in patients with exaggerated tissue inflammatory responses can have favourable patient outcome.

Galectin-3 deficiency drives lupus-like disease by promoting spontaneous germinal centers formation via IFN-\u03b3

Germinal centers (GC) are important sites for high-affinity and long-lived antibody induction. Tight regulation of GC responses is critical for maintaining self-tolerance. Here, we show that Galectin-3 (Gal-3) is involved in GC development. Compared with WT mice, Gal-3 KO mice have more GC B cells and T follicular helper cells, increased percentages of antibody-secreting cells and higher concentrations of immunoglobulins and IFN-γ in serum, and develop a lupus-like disease. IFN-γ blockade in Gal-3 KO mice reduces spontaneous GC formation, class-switch recombination, autoantibody production and renal pathology, demonstrating that IFN-γ overproduction sustains autoimmunity. The results from chimeric mice show that intrinsic Gal-3 signaling in B cells controls spontaneous GC formation. Taken together, our data provide evidence that Gal-3 acts directly on B cells to regulate GC responses via IFN-γ and implicate the potential of Gal-3 as a therapeutic target in autoimmunity.

CD8+ T cells in beige adipogenesis and energy homeostasis.

Although accumulation of lymphocytes in the white adipose tissue (WAT) in obesity is linked to insulin resistance, it remains unclear whether lymphocytes also participate in the regulation of energy homeostasis in the WAT. Here, we demonstrate enhanced energy dissipation in Rag1-/- mice, increased catecholaminergic input to subcutaneous WAT, and significant beige adipogenesis. Adoptive transfer experiments demonstrated that CD8+ T cell deficiency accounts for the enhanced beige adipogenesis in Rag1-/- mice. Consistently, we identified that CD8-/- mice also presented with enhanced beige adipogenesis. The inhibitory effect of CD8+ T cells on beige adipogenesis was reversed by blockade of IFN-γ. All together, our findings identify an effect of CD8+ T cells in regulating energy dissipation in lean WAT, mediated by IFN-γ modulation of the abundance of resident immune cells and of local catecholaminergic activity. Our results provide a plausible explanation for the clinical signs of metabolic dysfunction in diseases characterized by altered CD8+ T cell abundance and suggest targeting of CD8+ T cells as a promising therapeutic approach for obesity and other diseases with altered energy homeostasis.

CD8 T Cell Memory Increases Immunopathology in the Perforin-Deficient Model of Hemophagocytic Lymphohistiocytosis Secondary to TNF-α.

Familial hemophagocytic lymphohistiocytosis 2 (FHL2) is a cytokine storm syndrome characterized by immune hyperactivation with viral infection due to a CD8 T cell cytotoxic killing defect secondary to a perforin deficiency. As most studies of FHL2 mice have used pathogen naïve animals, the effects of immune memory on FHL2 are understudied. We utilized an immunization model of the perforin-deficient mouse to study the effects of immune memory on FHL2. Prior CD8 T cell specific antigen exposure leads to enhanced HLH disease with increased morbidity and decreased time to mortality. Enhanced disease is associated with altered cytokine production and T cell proliferation. Response to IFNγ blockade is reduced and TNFα gains a pathogenic role, while blockade of the IL-33 receptor ST2 remains effective. These results suggest that pre-existing immune memory may worsen outcome and alter treatment response for FHL2 patients who may not be naïve to their immune triggers.

P064 INTERFERON-GAMMA INDUCED VASCULAR IMPAIRMENT CONTRIBUTES TO THE PATHOGENESIS OF INFLAMMATORY BOWEL DISEASES

Inflammatory Bowel Diseases (IBD) are characterized by upregulation of Interferon-gamma (IFN-γ). Recently we showed that IFN-γ blockade by a specific antibody in Dextran Sodium Sulfate (DSS)-colitis mouse model results in increased angiogenesis and reduced vessel permeability(1). The aim of the present project was to validate the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD. For examination of vascular IFN-γ effects in vivo, we established an endothelial cell-specific Ifngr2-knock-out (Ifngr2ΔEndoC) mouse model. Vessel sprouting after IFN-γ stimulation of metatarsals was used for functional validation of the knock out. In order to analyze the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD, Ifngr2ΔEndoC and control mice were subjected to DSS-colitis. Angiogenesis and vascular barrier function of the colon were compared. Comparison of metatarsal angiogenic sprouting from Ifngr2ΔEndoC and control mice showed that endothelial-specific knock-out of Ifngr2 abrogates the angiostatic effects of IFN-γ. Induction of DSS-colitis revealed that Ifngr2ΔEndoC mice, compared to control mice, had a reduced inflammation of the colon. Investigation of specific vascular alterations showed an increased angiogenesis rate for Ifngr2ΔEndoC mice accompanied by an improved vascular barrier function, marked by higher vascular pericyte coverage and reduced vessel permeability. Our results indicate for first time an important pathofunction of the blood vessels in IBD. Specifically an IFN-γ-induced disruption of the vascular barrier function exaggerated the course of disease. This may open new therapy options. 1. Haep et al. Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice. Inflamm Bowel Dis. 2015.

Bidirectional modulation between infiltrating CD3+ T-lymphocytes and astrocytes in the spinal cord drives the development of allodynia in monoarthritic rats

Increasing evidence suggests that T cells and glia participate in the process of neuropathic pain. However, little is known about the involvement of T cells or the interaction between glia and T cells at the molecular level. Here we investigated the phenotype of T cell infiltration into the spinal cord in inflammatory pain and explored potential crosstalk between glia and T cells. The establishment of monoarthritis produced T cell infiltration and astrocyte activation, exhibiting similar kinetics in the spinal cord. T-cell-deficient (Rag1−/−) mice significantly attenuated MA-induced mechanical allodynia and GFAP upregulation. Double immunofluorescence staining showed that CD3 mainly colocalized with interferon-gamma (IFN-γ). Western blot and flow cytometry showed that multiple intrathecal administrations of astrocytic inhibitor fluorocitrate decreased IFN-γ-production without decreasing T cell number in the spinal cord. Spinal IFN-γ blockade reduced MA-induced mechanical allodynia and astroglial activation. In contrast, treatment with rIFN-γ directly elicited persistent mechanical allodynia and upregulation of GFAP and pJNK1/2 in naïve rats. Furthermore, rIFN-γ upregulated the phosphorylation of NF-κB p65 in cultured astrocytes vitro and spinal dorsal horn vivo. The results suggest that Th1 cells and astrocytes maintain inflammatory pain and imply that there may be a positive feedback loop between these cells via IFN-γ.

P064 INTERFERON-GAMMA INDUCED VASCULAR IMPAIRMENT CONTRIBUTES TO THE PATHOGENESIS OF INFLAMMATORY BOWEL DISEASES

Inflammatory Bowel Diseases (IBD) are characterized by upregulation of Interferon-gamma (IFN-γ). Recently we showed that IFN-γ blockade by a specific antibody in Dextran Sodium Sulfate (DSS)-colitis mouse model results in increased angiogenesis and reduced vessel permeability(1). The aim of the present project was to validate the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD. For examination of vascular IFN-γ effects in vivo, we established an endothelial cell-specific Ifngr2-knock-out (Ifngr2ΔEndoC) mouse model. Vessel sprouting after IFN-γ stimulation of metatarsals was used for functional validation of the knock out. In order to analyze the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD, Ifngr2ΔEndoC and control mice were subjected to DSS-colitis. Angiogenesis and vascular barrier function of the colon were compared. Comparison of metatarsal angiogenic sprouting from Ifngr2ΔEndoC and control mice showed that endothelial-specific knock-out of Ifngr2 abrogates the angiostatic effects of IFN-γ. Induction of DSS-colitis revealed that Ifngr2ΔEndoC mice, compared to control mice, had a reduced inflammation of the colon. Investigation of specific vascular alterations showed an increased angiogenesis rate for Ifngr2ΔEndoC mice accompanied by an improved vascular barrier function, marked by higher vascular pericyte coverage and reduced vessel permeability. Our results indicate for first time an important pathofunction of the blood vessels in IBD. Specifically an IFN-γ-induced disruption of the vascular barrier function exaggerated the course of disease. This may open new therapy options.

Tumor Necrosis Factor-α in Target Cells but Not in Effector Cells Plays Critical Roles in Development of Murine Immune-Mediated Bone Marrow Failure

Tumor necrosis factor alpha (TNF-α) is a critical cytokine in systemic inflammation and a potent inducer of apoptotic cell death. Type 1 cytokines are involved in the pathogenesis of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. Overproduction of interferon gamma (IFN-γ) and TNF-α from activated T cells or bone marrow cells has been proposed as a mechanism of BM failure. We hypothesized that inhibiting the production of TNF-α or blockade of the TNF-α signaling pathway would attenuate hematopoietic cell destruction in murine models of immune BM failure. Therefore, we tested the role of TNF-α in several mouse models of human AA, using C57BL/6 (B6) mice carrying germline deletions of TNF-α (TNF-α-/-) or its receptors including TNF-α receptor superfamily member 1a (TNF-rsf1a-/-), TNF-rsf1b-/-, and TNF-rsf1a-/-1b-/-as donors or recipients, respectively.Lymph node (LN) cells from both TNF-α-/- and TNF-α receptor knock out mice induced fatal BM failure with efficacy similar to LN cells from wild-type B6 donors when infused into CByB6F1 recipients that had received 5 Gy of total body irradiation (TBI), indicating that expression of TNF-α or TNF-α receptors on effector cells did not affect the development of BM failure.However, TNF-α-/- mice as recipients unexpectedly showed resistance to immune-mediated BM failure. Infusion of 5 million LN cells from MHC-mismatched FVB donors to 6.5 Gy TBI pre-conditioned TNF-α-/- recipients did not induce AA: the animals had normal or increased levels of WBC, RBC, platelets, and total BM cells relative to TBI-only TNF-α-/- mice; control wild-type B6 recipient mice developed severe pancytopenia and BM failure after LN infusion at 10 days (Fig. 1A&B). This result suggests that TNF-α production in the recipients had a role in immune attack by effector cells. When residual BM cells were cultured in vitro in cytokine-supplemented methylcellulose media, cells from wild-type BM failure mice, but not from TNF-α-/- recipient mice, showed a significant reduction in colony forming units relative to their TBI controls. By flow cytometry, Fas+ cells and Annexin V-binding cells were increased in BM of wild-type marrow failure mice relative to wild-type TBI controls, but no increase in Fas+ cells and Annexin V-binding cells was observed in TNF-α-/- mice that had received LN infusion. In parallel experiments, injection of FVB LN cells into sub-lethally-irradiated TNF-rsf1a-/-, TNF-rsf1b-/-, or TNF-rsf1a-/-1b-/- mice resulted in BM failure with declines in WBC and platelets as observed in wild-type B6 mice after FVB LN infusion. Plasma levels of both IFN-γ and TNF-α were markedly increased in all the wild-type B6 and TNF-α receptor knock out mice that developed BM failure, but not in TNF-α-/- recipient mice. These data suggest that depletion of TNF-α receptors could not attenuate immune-mediated BM destruction and that TNF-α did not exert its destructive effect on hematopooetic targets through the TNF-α/TNF-rsf1a and 1b pathways.We hypothesized that TNF-α might modulate T cell IFN-γ production to affect immune-mediated BM destruction. TNF-α is mainly produced by activated macrophages, while IFN-γ is mainly secreted by activated T cells. Blockade of either IFN-γ in effector cells or IFN-γ receptor in target cells ameliorates the development of BM failure (Chen J, Blood.2015;126:2621). To test this hypothesis, we first depleted macrophages from CByB6F1 recipient mice with liposomal clodronate before BM failure induction with TBI and B6 LN cell injection. Indeed, removal of macrophages attenuated BM destruction relative to BM failure mice pre-injected with PBS-loaded liposomes without macrophage depletion (Figure 1C). When we injected recombinant TNF-α daily i.v. for 1 week into TNF-α-/- recipients following TBI and FVB LN cell infusion, TNF-α-injected mice had decreased BM cellularity relative to mice that received TBI and LN infusion without TNF-α (Figure 1D). Remarkably, TNF-α injection increased intracellular IFN-γ expression in BM T cells as determined by flow cytometry, indicating that TNF-α was capable of stimulating T cells to secrete IFN-γ.In summary, our study reveals a critical role of TNF-α in target cells but not in effector cells, in the pathogenesis of immune-mediated BM failure by cell-cell “crosstalk” modulation of IFN-γ secretion. [Display omitted] DisclosuresYoung:Novartis/GSK to institute: Research Funding.

Ebi3 Prevents Trypanosoma cruzi-Induced Myocarditis by Dampening IFN-γ-Driven Inflammation.

The identification of anti-inflammatory mediators can reveal important targetable molecules capable of counterbalancing Trypanosoma cruzi-induced myocarditis. Composed of Ebi3 and IL-27p28 subunits, IL-27 is produced by myeloid cells and is able to suppress inflammation by inducing IL-10-producing Tr1 cells, thus emerging as a potential candidate to ameliorate cardiac inflammation induced by T. cruzi. Although IL-27 has been extensively characterized as a suppressive cytokine that prevents liver immunopathogenesis after T. cruzi infection, the mechanisms underlying its effects on T. cruzi-induced myocarditis remain largely unknown. Here, wild-type (WT) and Ebi3-deficient animals were intraperitoneally infected with trypomastigotes of T. cruzi Y strain and used to evaluate the potential anti-inflammatory properties of Ebi3 during T. cruzi infection. The survival rates of mice were daily recorded, the frequency of inflammatory cells was analyzed by flow cytometry and inflammatory mediators were measured by ELISA, real-time PCR and PCR array. We reported that T. cruzi-induced myocarditis was prevented by Ebi3. Stressors mainly recognized by TLR2 and TLR4 receptors on myeloid cells were essential to trigger IL-27p28 production. In addition, Ebi3 regulated IFN-γ-mediated myocarditis by promoting an anti-inflammatory environment through IL-10, which was most likely produced by Tr1 cells rather than classical regulatory T cells (Tregs), in the heart tissue of T. cruzi-infected animals. Furthermore, in vivo IFN-γ blockade ameliorated the host survival without compromising the parasite control in the bloodstream. In humans, IL-27p28 was correlated with cardiac protection during Chagas disease. Patients with mild clinical forms of the disease produced high levels of IL-27p28, whereas lower levels were found in those with severe forms. In addition, polymorphic sites at Ebi3 gene were associated with severe cardiomyopathy in patients with Chagas disease. Collectively, we describe a novel regulatory mechanism where Ebi3 dampens cardiac inflammation by modulating the overproduction of IFN-γ, the bona fide culprit of Chagas disease cardiomyopathy.

Safety, pharmacokinetics and pharmacodynamics of AMG 811, an anti-interferon-γ monoclonal antibody, in SLE subjects without or with lupus nephritis

ObjectiveTo evaluate safety, pharmacokinetics and pharmacodynamics of anti-interferon (IFN)-γ monoclonal antibody AMG 811 in subjects with SLE without or with lupus nephritis (LN).MethodsIn this phase Ib, randomised, multiple-dose escalation study...

Interferon-γ-Driven iNOS: A Molecular Pathway to Terminal Shock in Arenavirus Hemorrhagic Fever

Arenaviruses such as Lassa virus (LASV) cause hemorrhagic fever. Terminal shock is associated with a systemic cytokine storm, but the mechanisms are ill defined. Here we used HLA-A2-expressing mice infected with a monkey-pathogenic strain of lymphocytic choriomeningitis virus (LCMV-WE), a close relative of LASV, to investigate the pathophysiology of arenavirus hemorrhagic fever (AHF). AHF manifested as pleural effusions, edematous skin swelling, and serum albumin loss, culminating in hypovolemic shock. A characteristic cytokine storm included numerous pro-inflammatory cytokines and nitric oxide (NO) metabolites. Edema formation and terminal shock were abrogated in mice lacking inducible nitric oxide synthase (iNOS), although the cytokine storm persisted. iNOS was upregulated in the liver inaTcell- and interferon-γ (IFN-γ)-dependent fashion. Accordingly, blockade of IFN-γ or depletion of Tcells repressed hepatic iNOS and prevented disease despite unchecked high-level viremia. We identify the IFN-γ-iNOS axis as an essential and potentially druggable molecular pathway to AHF-induced shock.

Chitosan oligosaccharide/Pluronic polymers nano-aggregates as a potential oral drug delivery system

Interferon-γ (IFN-γ) has been implicated in the pathogenesis of multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). The type-1 cannabinoid receptors (CB1Rs) are heavily involved in MS pathophysiology, and a growing body of evidence suggests that mood disturbances reflect specific effects of proinflammatory cytokines on neuronal activity. Here, we investigated whether IFN-γ could exert a role in the anxiety- and depressive-like behavior observed in mice with EAE, and in the modulation of CB1Rs. Anxiety and depression in fact are often diagnosed in MS, and have already been shown to depend on cannabinoid system. We performed biochemical, behavioral and electrophysiological experiments to assess the role of IFN-γ on mood control and on synaptic transmission in mice. Intracerebroventricular delivery of IFN-γ caused a depressive- and anxiety-like behavior in mice, associated with the selective dysfunction of CB1Rs controlling GABA transmission in the striatum. EAE induction was associated with increased striatal expression of IFN-γ, and with CB1R transmission deficits, which were rescued by pharmacological blockade of IFN-γ. IFN-γ was unable to replicate the effects of EAE on excitatory and inhibitory transmission in the striatum, but mimicked the effects of EAE on CB1R function in this brain area. Overall these results indicate that IFN-γ exerts a relevant control on mood, through the modulation of CB1R function. A better understanding of the biological pathways underling the psychological disorders during neuroinflammatory conditions is crucial for developing effective therapeutic strategies.

Interferon-γ causes mood abnormalities by altering cannabinoid CB1 receptor function in the mouse striatum

Interferon-γ (IFN-γ) has been implicated in the pathogenesis of multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). The type-1 cannabinoid receptors (CB1Rs) are heavily involved in MS pathophysiology, and a growing body of evidence suggests that mood disturbances reflect specific effects of proinflammatory cytokines on neuronal activity. Here, we investigated whether IFN-γ could exert a role in the anxiety- and depressive-like behavior observed in mice with EAE, and in the modulation of CB1Rs. Anxiety and depression in fact are often diagnosed in MS, and have already been shown to depend on cannabinoid system. We performed biochemical, behavioral and electrophysiological experiments to assess the role of IFN-γ on mood control and on synaptic transmission in mice. Intracerebroventricular delivery of IFN-γ caused a depressive- and anxiety-like behavior in mice, associated with the selective dysfunction of CB1Rs controlling GABA transmission in the striatum. EAE induction was associated with increased striatal expression of IFN-γ, and with CB1R transmission deficits, which were rescued by pharmacological blockade of IFN-γ. IFN-γ was unable to replicate the effects of EAE on excitatory and inhibitory transmission in the striatum, but mimicked the effects of EAE on CB1R function in this brain area. Overall these results indicate that IFN-γ exerts a relevant control on mood, through the modulation of CB1R function. A better understanding of the biological pathways underling the psychological disorders during neuroinflammatory conditions is crucial for developing effective therapeutic strategies.

Dampened inflammation with ruxolitinib treatment leads to CD8+ T cell exhaustion and induction of T regulatory cells in a murine model of HLH

Abstract Hemophagocytic lymphohistiocytoses (HLH) is a heterogeneous group of disorders characterized by severely damaging immune responses. In HLH, activated CD8+ T cells and macrophages overproduce pro-inflammatory cytokines, which contribute greatly to morbidity and mortality. In a murine preclinical model, HLH is recapitulated by infecting perforin knockout mice with lymphocytic choriomeningitis virus (LCMV). The inability of CD8+ T cells to kill infected cells results in a high production of inflammatory cytokines and ultimately death. We recently reported that treatment with the JAK1/2 inhibitor ruxolitinib significantly reduces the manifestations of HLH in this model. In this study, we sought to: 1) compare the effect of ruxolitinib treatment with IFN-γ blockade (currently in clinical trials) in the acute phase and 2) determine the long-term effects of treatment upon discontinuation. On day 9 p.i., ruxolitinib treatment was superior to IFN-γ blockade in reducing splenomegaly, numbers of inflammatory foci in the liver, LCMV-specific hepatic CD8+ T cells, and splenic IFN-γ+TNF-α+ CD8+ T cells. Surprisingly, on day 35 p.i., ruxolitinib treated mice achieved 100% survival, while only 20% of the IFN-γ Ab-treated mice survived. Since ruxolitinib dampens inflammation, we hypothesized that lower inflammation and viral persistence would induce T cell exhaustion. Indeed, CD8+ T cells stimulated with LCMV-specific peptide produced less IFN-γ and TNF-α. In addition, a significant population of CD8+CD44hi T cells co-expressed PD-1 and Tim-3. This was also correlated with enhanced percentages of Foxp3+CD4+ Tregs in treated mice. Overall, this study provides evidence and mechanistic insight for ruxolitinib as a novel treatment for HLH.

Chronic Toxoplasma gondii Infection Exacerbates Secondary Polymicrobial Sepsis

Sepsis is a severe syndrome that arises when the host response to an insult is exacerbated, leading to organ failure and frequently to death. How a chronic infection that causes a prolonged Th1 expansion affects the course of sepsis is unknown. In this study, we showed that mice chronically infected with Toxoplasma gondii were more susceptible to sepsis induced by cecal ligation and puncture (CLP). Although T. gondii-infected mice exhibited efficient control of the bacterial burden, they showed increased mortality compared to the control groups. Mechanistically, chronic T. gondii infection induces the suppression of Th2 lymphocytes via Gata3-repressive methylation and simultaneously induces long-lived IFN-γ-producing CD4+ T lymphocytes, which promotes systemic inflammation that is harmful during CLP. Chronic T. gondii infection intensifies local and systemic Th1 cytokines as well as nitric oxide production, which reduces systolic and diastolic arterial blood pressures after sepsis induction, thus predisposing the host to septic shock. Blockade of IFN-γ prevented arterial hypotension and prolonged the host lifespan by reducing the cytokine storm. Interestingly, these data mirrored our observation in septic patients, in which sepsis severity was positively correlated to increased levels of IFN-γ in patients who were serologically positive for T. gondii. Collectively, these data demonstrated that chronic infection with T. gondii is a critical factor for sepsis severity that needs to be considered when designing strategies to prevent and control the outcome of this devastating disease.

Proangiogenic Function of T Cells in Corneal Transplantation.

Corneal neovascularization increases the risk of T cell-mediated allograft rejection. Here, we investigate whether T cells promote angiogenesis in transplantation. Conventional effector T cells were collected from draining lymph nodes of allogeneic or syngeneic corneal transplanted BALB/c mice. T cells were either cocultured with vascular endothelial cells (VECs) to assess VEC proliferation or used in a mixed lymphocyte reaction assay. Messenger RNA (mRNA) expression of vascular endothelial growth factor (VEGF)-A, -C, and VEGF receptor 2 (VEGF-R2) in VECs was assessed by real-time PCR. VEGF-A protein expression was determined by enzyme-linked immunosorbent assay. Flow cytometry was used to analyze VEGF-R2 expression in corneal CD31 cells, and VEGF-A and IFNγ expression in corneal CD4 T cells. Allogeneic T cells from high-risk (HR) grafted mice induced more VEC proliferation than those from syngeneic transplant recipients (P = 0.03). Vascular endothelial growth factor-A mRNA and protein expression were higher in T cells from draining lymph nodes (P = 0.03 and P = 0.04, respectively) and cornea (protein; P = 0.04) of HR compared with low-risk (LR) grafted hosts. Vascular endothelial growth factor-A, VEGF-C, and VEGF-R2 mRNA expression were increased in VECs when cocultured with T cells from HR transplants compared with LR transplants and naive mice. In addition, IFNγ blockade in T cell/VEC coculture increased VEC proliferation and VEGF-A protein expression, whereas blocking VEGF-A significantly reduced VEC proliferation (P = 0.04). Allogeneic T cells from corneal transplant hosts promote VEC proliferation, probably via VEGF-A signaling, whereas IFNγ shows an antiangiogenic effect. Our data suggest that T cells are critical mediators of angiogenesis in transplantation.

Abstract B34: Melanoma cells respond to CD8+ T cell attack by upregulation of genes associated with antigen processing and presentation

Abstract T cell-based immunotherapies have brought great progress for cancer patients, but many questions still remain open with regard to mechanisms of action and immune regulation in the tumor microenvironment. Methods: We setup a well-controlled co-culture system to study the dynamic T cell - cancer cell interplay. Low passage MelanA-expressing melanoma cell lines were cultured with MelanA-specific CD8+ T cells and characterized using differential gene expression analysis and flow cytometry. Results: As expected, significant fractions of melanoma cells died in presence of melanoma-specific CD8+ T cells. However, still some melanoma cells persisted during up to three days of co-culture. Characterization of the surviving melanoma cells revealed increased mRNA levels of genes associated with antigen processing and presentation: HLA Class I-encoding genes (HLA-A and HLA-B), HLA Class 2-encoding genes (HLA-DRA and HLA-DRB) and CD74, the invariant chain that stabilizes the complex of HLA Class II α and β chains until it is loaded with a peptide, were strongly increased after co-culture, as well as TAP1 and TAP2 (encoding transporter associated with antigen presentation), responsible for peptide transfer from the cytosol to the endoplasmic reticulum (Neefjes et al., 2011). HLA-Class I and HLA-DR expression was also strongly increased at the protein level as assessed at 48h of co-culture. These gene and protein expression changes were dependent on antigen-specific interaction of CTLs with melanoma cells and were mediated by IFNγ and TNFα, two cytokines secreted by CTLs upon antigen recognition. Discussion: So far, mainly immunosuppressive mechanisms of cancer cells upon exposure to CTLs were described, such as increased expression of IDO1 and the inhibitory receptor ligand PDL1 (Spranger et al., 2013). Here we show that further to these immunosuppressive mechanisms, melanoma cells respond to melanoma-specific CTLs with an IFNγ-driven upregulation of genes involved in antigen presentation. Increased antigen-presentation likely increases target cell recognition of CTLs. These findings may play a role in successful immunotherapy, and might explain why IFNγ blockade in situations of anti-tumor immune attack not only blocks the immunosuppressive response of melanoma cells but also the anti-tumor T cell response (McGray et al., 2013).

Combined Treatment with Ruxolitinib and Dexamethasone Curtails Inflammation and Lessens Disease in Preclinical Studies of Hemophagocytic Lymphohistiocytosis

The Hemophagocytic lymphohistiocytoses (HLH) comprise a heterogeneous group of disorders characterized by abnormal and severely damaging immune responses. Patients with HLH experience episodes of inflammation associated with the hyper-activation of CD8+ T cells and macrophages, which overproduce pro-inflammatory cytokines including interferon (IFN)-γ, interleukins (IL)-12 and -18, -6 and Tumor Necrosis Factor-α. Current treatments include immunosuppressive medications (corticosteroids, anti-thymocyte globulin) with or without cytotoxic chemotherapeutic agents (etoposide). Despite the use of these drugs, >50% of HLH patients die from unbridled inflammation. To develop more effective treatments for HLH, our laboratory has focused on blocking the effects of cytokines, which drive inflammation and mediate morbidity and mortality in HLH. We recently demonstrated that targeting the Janus Kinases (JAK1/2) with the JAK inhibitor ruxolitinib (INCB018424) significantly ameliorates the clinical and laboratory manifestations of HLH in preclinical mouse models (R. Das et al., Blood, 2016). Despite its beneficial effects, ruxolitinib did not completely abrogate disease. We therefore sought to understand its mechanisms of action and further improve upon these results. In mice, IFN-γ is central to disease pathogenesis, as its neutralization reduces anemia and prolongs survival. To examine whether ruxolitinib might be acting primarily through the inhibition of this cytokine, we modeled HLH in perforin-deficient (Prf1-/-) mice that were infected with lymphocytic choriomeningitis virus (LCMV). Beginning on day 4 post infection (p.i.), mice were treated or not with ruxolitinib (90 mg/kg by oral gavage twice daily) or with an IFN-γ blocking antibody (0.5 mg intraperitoneally [i.p.] on days 4 and 7). On day 9 p.i., the end point for these studies, mice were examined for the manifestations of HLH. Compared to untreated LCMV-infected mice, which lost on average 16% of starting body weight, mice treated with ruxolitinib or IFN-γ blockade exhibited only 7% weight loss. Ruxolitinib and IFN-γ blockade also ameliorated LCMV-induced anemia and thrombocytopenia, with platelet counts with ruxolitinib treatment reaching levels comparable to those observed in uninfected animals. Of note, ruxolitinib treatment decreased splenomegaly by over 50% (p=0.0001), while IFN-γ blockade had no effect. When compared to mice that received either no treatment or IFN-γ blockade, mice treated with ruxolitinib also exhibited a reduction in inflammation, which was manifested as a significantly lower area of liver encompassed by inflammatory foci, as well as a marked reduction in the absolute number of total and gp33-tetramer reactive splenic and intrahepatic CD8+ T cells. Together, these results suggest that ruxolitinib exerts its effects through mechanisms that are in part independent of the inhibition of IFN-γ signaling. We next examined whether combining ruxolitinib with dexamethasone, a gold standard medication in HLH treatment, might further diminish inflammation. To this end, LCMV-infected Prf1-/- mice were treated or not with ruxolitinib (as above), dexamethasone (15mg/kg i.p. daily) or ruxolitinib and dexamethasone. All treatments resulted in a similar reduction in weight loss. Thrombocytopenia was abrogated with ruxolitinib treatment, either alone or in combination with dexamethasone. Mice treated with both drugs, however, did not develop splenomegaly and were less anemic than mice treated with either single agent. Strikingly, when compared to the livers of infected untreated mice, livers from mice treated with combination therapy exhibited a 99.7% decrease in the area encompassed by inflammatory foci. This was in contrast to mice treated with ruxolitinib or dexamethasone, which showed a 66.8% or 87% reduction, respectively. Consistent with these findings, combination therapy was also more potent in decreasing the numbers and to a lesser extent the frequencies of total and antigen-specific CD8+ T cells. These findings suggest that treatment with ruxolitinib and dexamethasone further limits inflammation and lessens the manifestations of HLH, which represents a serious clinical problem for which there has been limited progress in treatment. The results of these studies support the incorporation of ruxolitinib and dexamethasone into future clinical trials to improve the cure rate for HLH. DisclosuresNo relevant conflicts of interest to declare.