IFN-gamma Receptor Research Articles

Reactogenicity and immunogenicity of the second COVID-19 vaccination in patients with inborn errors of immunity or mannan-binding lectin deficiency.

BackgroundPatients with inborn errors of immunity (IEI) are at increased risk for severe courses of SARS-CoV-2 infection. COVID-19 vaccination provides effective protection in healthy individuals. However, it remains unclear whether vaccination is efficient and safe in patients with constitutional dysfunctions of the immune system. Thus, we analyzed the humoral response, adverse reactions and assessed the disease activity of the underlying disease after COVID-19 vaccination in a cohort of patients suffering from IEIs or mannan-binding lectin deficiency (MBLdef).MethodsVaccination response was assessed after basic immunization using the Elecsys anti-SARS-CoV-2 S immunoassay and via Vero E6 cell based assay to detect neutralization capabilities. Phenotyping of lymphocytes was performed by flow cytometry. Patient charts were reviewed for disease activity, autoimmune phenomena as well as immunization status and reactogenicity of the vaccination. Activity of the underlying disease was assessed using a patient global numeric rating scale (NRS).ResultsOur cohort included 11 individuals with common variable immunodeficiency (CVID), one patient with warts hypogammaglobulinemia immunodeficiency myelokathexis (WHIM) syndrome, two patients with X-linked agammaglobulinemia (XLA), one patient with Muckle Wells syndrome, two patients with cryopyrin-associated periodic syndrome, one patient with Interferon-gamma (IFN-gamma) receptor defect, one patient with selective deficiency in pneumococcal antibody response combined with a low MBL level and seven patients with severe MBL deficiency. COVID-19 vaccination was generally well tolerated with little to no triggering of autoimmune phenomena. 20 out of 26 patients developed an adequate humoral vaccine response. 9 out of 11 patients developed a T cell response comparable to healthy control subjects. Tested immunoglobulin replacement therapy (IgRT) preparations contained Anti-SARS-CoV-2 S antibodies implicating additional protection through IgRT.SummaryIn summary the data support the efficacy and safety of a COVID-19 vaccination in patients with IEIs/MBLdef. We recommend evaluation of the humoral immune response and testing for virus neutralization after vaccination in this cohort.

Seq-ing the source of radiation-induced anti-tumor immunogenicity in patient tumors

Abstract Translational studies in a small number of patients have demonstrated a clinical benefit when combining radiation and immunotherapy. These findings are supported by correlative data showing that transcriptional changes to the tumor microenvironment following radiotherapy are associated with responses. Yet, given limited access to patient specimens following treatment, few studies have addressed how radiation primes anti-tumor immunity at single cell resolution, thus limiting our understanding of the source of radiation-induced changes within tumors. Here, we acquired renal cell carcinoma (RCC) tumors from patients treated with stereotactic body radiation therapy (SBRT) followed by nephrectomy in a pilot study (NCT01892930), to evaluate radiation-induced transcriptional changes to immune and RCC cells by single-cell RNA sequencing. We hypothesized that SBRT-induced interferon (IFN) and immune-activating signals, previously detected by bulk sequencing and array platforms, would localize to the dense lymphocyte infiltrate characterized in RCC and that the transcriptional signatures in SBRT-treated RCC would reflect increased immune recognition. Single-cell transcriptomics uncovered increased frequency of mature T cells in irradiated tumors, including increased expression of IFN-gamma and granzymes, as well as PD1 and other exhaustion markers. Additionally, irradiated-RCC cells expressed more IFN-gamma receptor and were enriched for IFN signaling and antigen processing pathways. Collectively, these results indicate radiation may improve anti-tumor immunity by increasing both T cell activity and tumor cell sensitivity to pro-inflammatory cytokines and cytolysis in human malignancies.

Association of TNF rs668920841 and INRA111 polymorphisms with caprine brucellosis: A case-control study of candidate genes involved in innate immunity

Caprine brucellosis is an infectious, contagious zoonotic disease caused by Brucella melitensis. Multiple factors, including host genetics, can influence the outcome of the exposure to Brucella; and it is expected that genetic variants that affect the host innate immune response could have a key role in Brucella infection and pathogenesis. In this study, we evaluated if polymorphisms in innate immunity-related genes are associated with results of Brucella infection in goats. Nine polymorphisms within interferon gamma (IFNG), tumor necrosis factor (TNF), MyD88 innate immune signal transduction adaptor (MYD88), interleukin 10 (IL10) and IL-10 receptor subunit alpha (IL10RA) genes and two molecular markers (BMS2753 and INRA111) were resolved by PCR-capillary electrophoresis in samples from 81 seronegative and 61 seropositive goats for brucellosis. A heterozygous genotype at INRA111, a microsatellite near the VRK serine/threonine kinase 2 (VRK2) gene, was associated with absence of Brucella-specific antibodies in goats naturally exposed to the pathogen (P = .004). Conversely, variants in the TNF gene (rs668920841) and near the IFN gamma receptor 1 (IFNGR1) gene (microsatellite BMS2753) were significantly associated with presence of Brucella-specific antibodies at allelic (P = .042 and P = .046) and genotypic level (P = .012 and P = .041, respectively). Moreover, an in silico analysis predicted a functional role of the insertion-deletion polymorphism rs668920841 on the transcriptional regulation of the caprine TNF gene. Altogether, these results contribute to the identification of genetic factors that have a putative effect on the resistance / susceptibility phenotype of goats to Brucella infection.

Autoantigen specific IL-2 activated CD4+CD25+T regulatory cells inhibit induction of experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4+CD25+T cells both from sensitized hosts or from naïve hosts after ex-vivo activation by PNM and rIL-2. Transfer of naïve CD4+CD25+T cells has no effect on EAN, nor did naïve CD4+CD25+T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4+CD25+Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naïve CD4+CD25+T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential.

Alloactivation of Naïve CD4+CD8-CD25+T Regulatory Cells: Expression of CD8α Identifies Potent Suppressor Cells That Can Promote Transplant Tolerance Induction.

Therapy with alloantigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rβ2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8−CD25+T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10–20% and CD8β on <5% of these cells. These cells expressed ifngr and Il12rb2. CD8α+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8α+CD25+Treg consistent with the CD8α+ cells expressing IL-12R. In MLC, the CD8α+ fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4+CD8−CD25+T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α+ cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4+CD8−CD25+Foxp3+T cells was a marker of activated and potent Treg that included alloantigen-specific Treg.

Recently alloactivated CD4+CD8−CD25+T regulatory cells express CD8alpha and are potent suppressor cells

Abstract Therapy with antigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are non-antigen specific and weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells and IL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rb2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined other markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8−CD25+T cells with rIL-2 and PVG alloantigen, CD8a was expressed on 10–20% and CD8b on &amp;lt;5% of these cells. RT-PCR showed the CD8+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8+CD25+Treg consistent with the CD8a+ cells expressing IL-12R. In MLC, the CD8+ fraction had greater suppression than the mixed Ts1 population, whereas the CD4+CD8−CD25+T cells had less suppression. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8a cells did not prevent rejection. This study showed the expression of CD8alpha by rIL-2 and alloantigen activation of CD4+CD8−CD25+Foxp3+T cells was a marker of alloantigen-specific Treg and may be of use to enrich alloantigen-specific Treg.

Cytokine, C-Reactive Protein, and Heat Shock Protein mRNA Expression Levels in Patients with Active Behçet's Uveitis.

BackgroundTo investigate the gene expression levels of interleukin 10 (IL10), IL18, interferon gamma (IFNG), IFN-gamma receptor (IFNGR), C-reactive protein (CRP), and heat shock protein 70 (HSP70) in patients with active Behçet’s uveitis.Material/MethodsForty patients with Behçet’s disease diagnosed according to the International Study Group criteria and 30 healthy individuals were included in the study. IL10, IL18, IFNG, IFNGR, CRP, and HSP70 gene expression levels were compared.ResultsExpression levels of IL18, IFNG, IFNGR, and CRP were significantly higher in patients with active Behçet’s uveitis than in control subjects (P<0.01 for all), whereas no significant differences were found in IL10 and HSP70 gene expression levels (P>0.01 for both).ConclusionsIL18, IFNG, IFNGR, and CRP gene expression is significantly increased in active Behçet’s uveitis. There was no significant difference between active Behçet’s uveitis patients and controls in terms of IL10 and HSP70 gene expression levels. We conclude that drugs prescribed to Behçet’s patients with active uveitis downregulate gene expression.

Post-translational modification of the interferon-gamma receptor alters its stability and signaling.

The IFN gamma receptor 1 (IFNGR1) binds IFN-γ and activates gene transcription pathways crucial for controlling bacterial and viral infections. Although decreases in IFNGR1 surface levels have been demonstrated to inhibit IFN-γ signaling, little is known regarding the molecular mechanisms controlling receptor stability. Here, we show in epithelial and monocytic cell lines that IFNGR1 displays K48 polyubiquitination, is proteasomally degraded, and harbors three ubiquitin acceptor sites at K277, K279, and K285. Inhibition of glycogen synthase kinase 3 beta (GSK3β) destabilized IFNGR1 while overexpression of GSK3β increased receptor stability. We identified critical serine and threonine residues juxtaposed to ubiquitin acceptor sites that impacted IFNGR1 stability. In CRISPR-Cas9 IFNGR1 generated knockout cell lines, cellular expression of IFNGR1 plasmids encoding ubiquitin acceptor site mutations demonstrated significantly impaired STAT1 phosphorylation and decreased STAT1-dependent gene induction. Thus, IFNGR1 undergoes rapid site-specific polyubiquitination, a process modulated by GSK3β. Ubiquitination appears to be necessary for efficient IFNGR1-dependent gamma gene induction and represents a relatively uncharacterized regulatory mechanism for this receptor.

Identification of a single nucleotide polymorphism indicative of high risk in acute myocardial infarction.

Background & objectives:Acute myocardial infarction (AMI) is a major health concern in India. The aim of the study was to identify single nucleotide polymorphisms (SNPs) associated with AMI in patients using dedicated chip and validating the identified SNPs on custom-designed chips using high-throughput microarray analysis.Methods:In pilot phase, 48 AMI patients and 48 healthy controls were screened for SNPs using human CVD55K BeadChip with 48,472 SNP probes on Illumina high-throughput microarray platform. The identified SNPs were validated by genotyping additional 160 patients and 179 controls using custom-made Illumina VeraCode GoldenGate Genotyping Assay. Analysis was carried out using PLINK software.Results:From the pilot phase, 98 SNPs present on 94 genes were identified with increased risk of AMI (odds ratio of 1.84-8.85, P=0.04861-0.003337). Five of these SNPs demonstrated association with AMI in the validation phase (P<0.05). Among these, one SNP rs9978223 on interferon gamma receptor 2 [IFNGR2, interferon (IFN)-gamma transducer 1] gene showed a significant association (P=0.00021) with AMI below Bonferroni corrected P value (P=0.00061). IFNGR2 is the second subunit of the receptor for IFN-gamma, an important cytokine in inflammatory reactions.Interpretation & conclusions:The study identified an SNP rs9978223 on IFNGR2 gene, associated with increased risk in AMI patient from India.

Expression of negative immune regulatory molecules, pro-inflammatory chemokine and cytokines in immunopathology of ECM developing mice

The pathological events in human cerebral malaria are mimicked in the experimental cerebral malaria (ECM) in Plasmodium berghei ANKA (PBA)-infected C57BL/6 mice. Although previously implied in ECM, the kinetics of cytokines and chemokines expression-an essential functional feature for defining causality in ECM development-remained untested. Herein, we characterized the immunopathological changes and the expression of negative immune regulatory molecules, cytokines and chemokines through asymptomatic (3days after infection, 3dpi), symptomatic (5dpi) and ECM (7dpi) stages in PBA-infected C57BL/6 mice. Parasitized RBCs were first detected in brain on 3dpi, edema and tissue alterations on 5dpi, and hemorrhages in different areas of brain on 7dpi. Increased cerebellar PD-1, CTLA-4 and LAG-3 expression and reduced hippocampal CXCL-4 expression on 3dpi were the first observed immunological changes. The negative immune regulatory molecules (PD-L1, CTLA-4), cytokines (TNF-α, sFAS-L), and chemokines (CXCL-10, MIP-1β) transcript levels varied in different brain areas in symptomatic and ECM phases. By 5dpi, TNF-α, CXCL10 and MIP-1β significantly increased in all brain parts studied; IL-1RA in whole brain, whereas CXCL4 reduced in hippocampus and cerebrum. By 7dpi, the hippocampal PD-1, CXCL4 and CTLA-4 expression decreased but the cerebral, cerebellar and hippocampal PD-L1 expression were elevated. TNF-α, CXCL10, MIP-1β, PD-1, CTLA-4 and PD-L1 expression were up-regulated in different brain areas. The TNFR2, IFN-gamma receptor, Lymphotoxin-β receptor and sFAS-L transcripts significantly increased in brain in ECM. Our data characterize key dynamic immunopathological changes in brain to imply relationship to ECM development.

A randomised double-blind placebo-controlled 12- week feasibility trial of methotrexate added to treatment as usual in early schizophrenia: study protocol for a randomised controlled trial.

BackgroundMethotrexate is a commonly used anti-inflammatory and immunosuppressive drug. There is growing evidence that inflammatory processes are involved in the pathogenesis of schizophrenia. In our recent randomised double-blind placebo-controlled clinical trial in Pakistan and Brazil, the addition of minocycline (antibiotic and anti-inflammatory drug) for 1 year to treatment as usual reduced negative symptoms and improved some cognitive measures. A meta-analysis of cytokine changes in the peripheral blood has identified IL-2, IFN-gamma, TNF-alpha and soluble IL-2 receptor as trait markers of schizophrenia because their levels were elevated during acute exacerbations and reduced in remission. This suggests immune activation and an inflammatory syndrome in schizophrenia. Based on the evidence of the strong anti-inflammatory properties of methotrexate, we propose that low-dose methotrexate may be an effective therapy in early schizophrenia.Methods/DesignThis is a double-blind placebo-controlled study of methotrexate added to treatment as usual for patients suffering from schizophrenia, schizoaffective disorder, psychosis not otherwise specified or schizophreniform disorder. This will be with 72 patients, 36 in each arm over 3 months. There will be screening, randomisation and follow-up visits. Full clinical assessments will be carried out at baseline, 2, 4, 8 and 12 weeks. Social and cognitive assessments will be carried out at baseline and 12 weeks. Methotrexate will be given at a dose of 10 mgs orally once a week for a 3-month period.DiscussionEvidence suggests inflammatory processes are involved in the pathogenesis of schizophrenia and anti-inflammatory treatments have shown to have some beneficial effects. Methotrexate is a known immunosuppressant and anti-inflammatory drug. The aim of this study is to establish the degree of improvement in positive and negative symptoms, as well as cognitive functioning with the addition of methotrexate to treatment as usual.ClinicalTrials.gov identifier: NCT02074319 (24 February 2014).

Mice Deficient in Interferon-Gamma or Interferon-Gamma Receptor 1 Have Distinct Inflammatory Responses to Acute Viral Encephalomyelitis

Interferon (IFN)-gamma is an important component of the immune response to viral infections that can have a role both in controlling virus replication and inducing inflammatory damage. To determine the role of IFN-gamma in fatal alphavirus encephalitis, we have compared the responses of wild type C57BL/6 (WTB6) mice with mice deficient in either IFN-gamma (GKO) or the alpha-chain of the IFN-gamma receptor (GRKO) after intranasal infection with a neuroadapted strain of sindbis virus. Mortalities of GKO and GRKO mice were similar to WTB6 mice. Both GKO and GRKO mice had delayed virus clearance from the brain and spinal cord, more infiltrating perforin+ cells and lower levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNAs than WTB6 mice. However, inflammation was more intense in GRKO mice than WTB6 or GKO mice with more infiltrating CD3+ T cells, greater expression of major histocompatibility complex-II and higher levels of interleukin-17A mRNA. Fibroblasts from GRKO embryos did not develop an antiviral response after treatment with IFN-gamma, but showed increases in TNF-alpha, IL-6, CXCL9 and CXCL10 mRNAs although these increases developed more slowly and were less intense than those of WTB6 fibroblasts. These data indicate that both GKO and GRKO mice fail to develop an IFN-gamma-mediated antiviral response, but differ in regulation of the inflammatory response to infection. Therefore, GKO and GRKO cannot be considered equivalent when assessing the role of IFN-gamma in CNS viral infections.

Abstract A83: Induction of PD-L1 expression by cytotoxic agents through activation of NF-kB signal

Abstract Recently, growing evidence has shown that the therapeutic efficacy of chemotherapy largely depends on the host immune system. We analyzed gene expression microarray dataset GSE13525 to examine how cytotoxic agents influence the immune system. Expression of programmed cell death 1 ligand 1 (PD-L1) was increased following carboplatin (CBDCA) treatment in the human ovarian cancer cell line 36M2 from GSE13525 (p&amp;lt;0.05). Previously, we reported PD-L1 expression is associated with poor prognosis in ovarian cancer (Hamanishi J, PNAS, 2007), and that PD-L1 promotes progression of ovarian cancer by inducing host immune tolerance through its receptor programmed cell death 1 (PD-1) on peripheral cytotoxic T lymphocytes (Abiko K, Clin Can Res, 2013). The aim of this study was to investigate the molecular mechanism of chemotherapy-induced PD-L1 expression in ovarian cancer cells and to examine if inhibition of the PD-L1/PD-1 signal increases the efficacy of chemotherapy in ovarian cancer. Pathway analysis of gene expression microarray data from clinical ovarian cancer samples collected before and after chemotherapy (GSE15622) showed that both paclitaxel (PTX) (n=20) and CBDCA (n=14) treatment activated the NF-kB signaling pathway (single-sample Gene Set Enrichment Analysis; p&amp;lt;0.01, respectively). We treated human ovarian cancer cell lines (OVCAR8, ovary1847, RMG-II and SKOV3) and mouse ovarian cancer cell lines (ID8 and HM-1) with CBDCA (100uM), PTX (10uM), gemcitabine (GEM: 120uM), liposomal doxorubicine (2ug/ml) or endoxan (7uM) for 24 hours. All of these cytotoxic agents increased NF-kB-p65 and PD-L1 protein expression detected by Western blotting in all of the cell lines tested. Increased expression of PD-L1 was also confirmed using flow cytometry in HM1 and ID8 cells treated with PTX or GEM (p&amp;lt;0.05, respectively). Following siRNA-mediated knockdown of NF-kB in HM1, ID8, ovary1847 and OVCAR8, induction of PD-L1 expression was drastically impaired by the cytotoxic agents PTX or GEM. This change was independent of IFN-gamma signaling, which is a well-known activator of PD-L1 expression, because IFN-gamma was not secreted from the treated cells, and because shRNA-mediated knock down of IFN-gamma receptor 1 also did not change PD-L1 expression. In order to determine if PD-L1 expression on ovarian cancer cells induced by cytotoxic agents could serve as a molecular target in ovarian cancer, we performed in vivo experiments. We performed lentiviral transduction into ID8 cells using PD-L1 cDNA (ID8-PDL1), vector control (ID8-control) and microRNA targeting PD-L1 mRNA (ID8-mirPDL1). Following intraperitoneal injection of these ID8 cells into syngeneic C57/BL6 mice, we injected PTX or vehicle intraperitoneally (6 groups, n=12 in each group). The ID8-PD-L1 mice without PTX showed the worst prognosis, while ID8-mirPDL1 mice with PTX showed the best prognosis (p&amp;lt;0.01). In an independent experiment using GEM instead of PTX, the ID8-mirPDL1 with GEM treatment again showed the best prognosis (p&amp;lt;0.01). In summary, we found chemotherapeutic agents upregulate PD-L1 expression through activation of NF-kB signaling. Immunotherapeutic targeting of the PD-L1/PD-1 signal combined with chemotherapy may be a promising treatment modality against ovarian cancer. Citation Format: Junzo Hamanishi, Noriomi Matsumura, Peng Jin, Kaoru Abiko, Naoki Horikawa, Ken Yamaguchi, Tsukasa Baba, Susan K. Murphy, Ikuo Konishi, Masaki Mandai. Induction of PD-L1 expression by cytotoxic agents through activation of NF-kB signal. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A83.

Ligand-independent interaction of the type I interferon receptor complex is necessary to observe its biological activity

Ectopic coexpression of the two chains of the Type I and Type III interferon (IFN) receptor complexes (IFN-αR1 and IFN-αR2c, or IFN-λR1 and IL-10R2) yielded sensitivity to IFN-alpha or IFN-lambda in only some cells. We found that IFN-αR1 and IFN-αR2c exhibit FRET only when expressed at equivalent and low levels. Expanded clonal cell lines expressing both IFN-αR1 and IFN-αR2c were sensitive to IFN-alpha only when IFN-αR1 and IFN-αR2c exhibited FRET in the absence of human IFN-alpha. Coexpression of RACK-1 or Jak1 enhanced the affinity of the interaction between IFN-αR1 and IFN-αR2c. Both IFN-αR1 and IFN-αR2c exhibited FRET with Jak1 and Tyk2. Together with data showing that disruption of the preassociation between the IFN-gamma receptor chains inhibited its biological activity, we propose that biologically active IFN receptors require ligand-independent juxtaposition of IFN receptor chains assisted by their associated cytosolic proteins.

P1-01-15: Do Serum Cytokines Predict Breast Cancer Behavior?

Abstract Introduction: The role of inflammation in breast cancer development and progression is poorly understood. The potential for serum cytokines to predict breast cancer behavior is an intriguing hypothesis, and data to date, has been conflicting. We sought to determine if serum inflammatory cytokines could predict clinicopathologic factors in breast cancer patients. Methods: Fifty-seven women with newly diagnosed breast cancer (stage 0–4) were accrued to this IRB-approved study. Serum cytokine levels were obtained in 50 of these women; this was the cohort of interest. All patients underwent surgical therapy, and log serum cytokine levels were correlated to final pathologic results. Results: The median patient age was 51 (range; 21–79) with a median tumor size was 19.5 mm (range; 2.3−95.0 mm). A variety of cytokines were evaluated. None were found to correlate with tumor size, lymph node status, presence of metastases, tumor grade, or lymphovascular invasion (all p's &amp;gt; .05). While most of the cytokines evaluated did not correlate with hormone receptor status, a significant correlation was seen between serum IFN-gamma and estrogen receptor (p = .011) and progesterone receptor (p = .013) status. No correlation was seen with her-2-neu status (p &amp;gt; .05). Conclusions: Serum cytokine levels do not seem to correlate with tumor size, lymph node status or grade. However, the strong correlation between IFN-gamma serum levels and hormone receptor status is hypothesis-generating and requires further study. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-15.

Severe adverse reactions after vaccination with Japanese BCG vaccine: a review

Japanese BCG vaccine has been admitted by the quality control of World Health Organization (WHO) as the safest BCG vaccine in the world. Even though, BCG, as a live bacterial vaccine, inevitably causes dissemination beyond vaccination site and regional lymph-nodes to various part of the body under certain special conditions. We tried to review the clinical features and immunological status of the cases in which "severe" adverse reactions had developed after vaccination with Japanese freeze-dried BCG vaccine prepared from BCG substrain Tokyo. "Severe" adverse reaction was arbitrarily defined as the adverse reactions of clinical significance developed beyond vaccination site and regional ipsilateral axillary lymph-nodes. By the extensive search of the literatures, 39 cases were identified since 1951 when vaccination with freeze-dried BCG vaccine became compulsory by the Tuberculosis Prevention Law in Japan. Incidence rate was calculated as 0.0182 cases per 100,000 vaccinations. Clinical manifestations of bone and joint were reported in 27 cases (multiple sites: 15 cases, single site: 12 cases), abnormalities in chest X-ray in 13 cases, skin manifestations in 17 cases, diseases in other sites or organs in 8 cases. Most of the cases had lesions in multiple organs. Among these 39 cases, 13 had been diagnosed to have some types of primary immunodeficiency (5 cases: chronic granulomatous disease (CGD); 4 cases: severe combined immunodeficiency (SCID); 4 cases: IFN-gamma receptor 1 deficiency). Further, unidentified defects in cellular immunity were reported in other 6 cases. Death was reported in 6 cases, but in two cases the causes of death were the infections due to different pathogens, namely, pulmonary abscess due to Staphylococcus sp. and bacteremia due to Pseudomonas aeruginosa, respectively, and in only one case death was evidenced as due to disseminated BCG infection by autopsy. All of 6 death cases had some type of immunodeficiency. Apart from fatal cases, outcome of "severe" adverse events were generally favorable and they were successfully treated by anti-tuberculosis therapy with or without surgical treatment for bone and joint lesions. Because the risk of "severe" adverse reactions is high among those of primary immunodeficiency, so it is advisable to avoid BCG vaccination during the first three months after birth when the detection of immunodeficiency is practically impossible.

IFN-gamma inhibits hepatic progenitor cell activation in chronic liver damage

Background & Aims: Hepatic progenitor cells (HPC) are thought to promote a periportal ductular reaction (DR) and contribute to periportal fibrosis in HCV- and non-alcholic steatohepatitis (NASH)-associated liver diseases. IFN-gamma is a well recognized fibrotic inhibitor in vitro and in vivo. The present study investigate the role of IFN-gamma in HPC activation in chronic HBV infected patients and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treated mouse. Methods: Hepatic progenitor cells were quantified by Cytokeratin-19 (CK19) immunostaining in 110 HBV, as were oval cells (HPCs in rodents) in DDC treated wild-type and three kinds of IFN-gamma-associated gene knockout mice, including IFN-gamma-, IFN-gamma receptor- and interferon regulator factor (IRF)-1 knockout mice. Results: The CK19 staining score significantly correlates with inflammatory grade (r=0.61, P<0.001) and fibrotic stage (r=0.61, P<0.001) in 110 chronic HBV infected patients. 9-months IFN-gamma treatment resulted in decreased HPC numbers concomitant with reduced inflammatory and fibrotic degree in chronic HBV infection (13/18 patients). In line with HBV patients, the number of oval cells and DRs significantly increased in IFN-gamma knockout mice compare with wild-type mice after 4 weeks DDC treatment. Furthermore, elevated oval cells and DR also demonstrated in interferon regulator factor (IRF)-1, downstream of IFN-gamma signalling, knockout mice with 4 weeks DDC administration. Either IFN-gamma- or IRF-1 knockout mice showed serious fibrotic degree compare with wild-type mice in DDC model. Conclusions: Besides a role in anti-fibrosis, IFN-gamma inhibits HPC activation in chronic liver damage.

The Immunoregulatory Enzyme Indoleamine 2,3-Dioxygenase (IDO1) Is Expressed by Natural Killer (NK) Cells During Cytokine-Mediated Activation

AbstractAbstract 3894 Introduction:human NK cells are large, granular cells derived from lymphocyte lineage. They are critical mediators of the innate immunity as they rapidly respond to pathogens infected and tumour cells through cytotoxic and cytokines-producing responses. Indoleamine 2,3-dioxygenase (IDO1) is an enzyme catalyzing the degradation of the essential amino acid L-tryptophan into kynurenines. Several cell types, including dendritic cells, have been shown to express IDO1, which acts as a potent immunosuppressive agent. Recently, in addiction to IDO1, the new variant IDO2 was described but its immunosuppressive role is still under investigations.The aim of the present work is to study the expression and the role of IDO1 and IDO2 in NK cells. Methods:CD3-CD56+NK cells were immunomagnetically purified from healthy donors. IFN-gamma production was measured by ELISA assay. IDO1 and DAP12 transcript levels were normalized on ABL. IDO2 expression was measured by semi-quantitative PCR. Kynurenine levels were evaluated by colorimetric assay. Results:peripheral blood NK cells were treated with IL-2 for 16h, 40h, 88h and 160h. At the end of each IL-2 stimulation, supernatants were collected and IFN-gamma production was measured by colorimetric assay. IDO1 expression in NK cells was evaluated by quantitative real-time PCR and Western blotting. Our data show that, during IL-2-mediated activation, IDO1 is up-regulated at mRNA and protein levels. This effect is maximum after an overnight incubation and it decreases at later time points. IDO1 expression is clearly correlated with IFN-gamma production by NK cells whereas IFN-gamma receptor expression is not affected. Culturing NK cells with a combination of IL-2 and anti-IFN-gamma antibody demonstrated that IDO1 expression is regulated, at least in part, by IFN-gamma. Interestingly, IDO1 mRNA upregulation is associated with downregulation of DAP12, a gene whose expression has been showed to be inversely correlated to IDO1 in dendritic cells. IDO2 expression seems to be not affected by IL-2 stimulation. Treatment with other inflammatory cytokines demonstrated that IDO1 is modulated by different stimuli. Overnight incubation with IL-12, IFN-gamma and a combination of IL-2 and IL-6 showed IDO1 induction. IFN-gamma showed the highest IDO1 induction at transcript and protein level and, surprisingly, of IDO2 expression.IDO1 expression by NK cells resulted in increased production of kynurenines and this effect was abrogated by the addition of the IDO1 inhibitor 1-methyl tryptophan. Conclusion:our results demonstrate that NK cells upregulate IDO1 expression during cytokine-mediated activation and that IDO1 expression is regulated, at least in part, by IFN-gamma. Similarly to DCs, NK cells express IDO1 gene in association with DAP12, thus suggesting a common regulatory pathway among different cell subsets. Disclosures:Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria.

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation

Autophagy is regulated for IFN-gamma-mediated antimicrobial efficacy; however, its molecular effects for IFN-gamma signaling are largely unknown. Here, we show that autophagy facilitates IFN-gamma-activated Jak2-STAT1. IFN-gamma induces autophagy in wild-type but not in autophagy protein 5 (Atg5(-/-))-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-gamma induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5(-/-) or Atg7(-/-) MEFs are, independent of changes in IFN-gamma receptor expression, resistant to IFN-gamma-activated Jak2-STAT1, which suggests that autophagy is important for IFN-gamma signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-gamma-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-gamma-induced activation of STAT1 in Atg5(-/-) MEFs. Our study provides evidence that there is a link between autophagy and both IFN-gamma signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.

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Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4+CD25+T cells both from sensitized hosts or from naïve hosts after ex-vivo activation by PNM and rIL-2. Transfer of naïve CD4+CD25+T cells has no effect on EAN, nor did naïve CD4+CD25+T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4+CD25+Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naïve CD4+CD25+T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential.