Spontaneous Interferon Research Articles

Whole genome deep sequencing analysis of viral quasispecies diversity and evolution in HBeAg seroconverters

SummaryBackground & AimsWe aimed to investigate how viral quasispecies of the HBV whole genome evolves and diversifies in response to HBeAg seroconversion and viral control utilising next-generation sequencing (NGS).MethodsFifty HBeAg-positive chronic hepatitis B patients, including 18 treatment-naïve and 32 interferon (IFN)-treated individuals, were recruited. Serial HBV whole genomes in serum were analysed by NGS to determine sequence characteristics and viral quasispecies.ResultsHBV quasispecies diversity, measured by nucleotide diversity, was negatively correlated with viral load and hepatitis activity. Spontaneous HBeAg seroconverters exhibited significantly greater viral quasispecies diversity than treatment-naïve non-seroconverters from >1 year before seroconversion (0.0112 vs. 0.0060, p <0.01) to >1 year after seroconversion (0.0103 vs. 0.0068, p <0.01). IFN-induced HBeAg seroconverters tended to have higher viral genetic diversity than non-seroconverters along with treatment. Particularly, the IFN responders, defined as IFN-induced HBeAg seroconversion with low viraemia, exhibited significantly greater genetic diversity of whole HBV genome at 6 months post-IFN treatment than IFN non-responders (0.0148 vs. 0.0106, p = 0.048). Moreover, spontaneous HBeAg seroconverters and IFN responders exhibited significantly higher evolutionary rates and more intra-host single-nucleotide variants. Interestingly, in spontaneous HBeAg seroconverters and IFN responders, there were distinct evolutionary patterns in the HBV genome.ConclusionsHigher HBV quasispecies diversity is associated with spontaneous HBeAg seroconversion and IFN-induced HBeAg seroconversion with low viraemia, conferring a favourable clinical outcome.Lay summaryHBeAg seroconversion is a landmark in the natural history of chronic HBV infection. Using next-generation sequencing, we found that the nucleotide diversity of HBV was negatively correlated with viral load and hepatitis activity. Patients undergoing HBeAg seroconversion had more diverse HBV genomes and a faster viral evolution rate. Our findings suggest HBeAg seroconversion is driven by host selection pressure, likely immune selection pressure.

CD8low T cells expanded following acute Trypanosoma cruzi infection and benznidazole treatment are a relevant subset of IFN-γ producers.

CD8 T cells are regarded as pivotal players in both immunoprotection and immunopathology following Trypanosoma cruzi infection. Previously, we demonstrated the expansion of CD8+ T lymphocytes in the spleen of T. cruzi-infected mice under treatment with benznidazole (N-benzyl-2-nitroimidazole acetamide; Bz), a drug available for clinical therapy. This finding underlies the concept that the beneficial effects of Bz on controlling acute T. cruzi infection are related to a synergistic process between intrinsic trypanocidal effect and indirect triggering of the active immune response. In the present study, we particularly investigated the effect of Bz treatment on the CD8+ T cell subset following T. cruzi infection. Herein we demonstrated that, during acute T. cruzi infection, Bz treatment reduces and abbreviates the parasitemia, but maintains elevated expansion of CD8+ T cells. Within this subset, a remarkable group of CD8low cells was found in both Bz-treated and non-treated infected mice. In Bz-treated mice, early pathogen control paralleled the lower frequency of recently activated CD8low cells, as ascertained by CD69 expression. However, the CD8low subset sustains significant levels of CD44highCD62Llow and CD62LlowT-bethigh effector memory T cells, in both Bz-treated and non-treated infected mice. These CD8low cells also comprise the main group of spontaneous interferon (IFN)-γ-producing CD8+ T cells. Interestingly, following in vitro anti-CD3/CD28 stimulation, CD8+ T cells from Bz-treated T. cruzi-infected mice exhibited higher frequency of IFN-γ+ cells, which bear mostly a CD8low phenotype. Altogether, our results point to the marked presence of CD8low T cells that arise during acute T. cruzi infection, with Bz treatment promoting their significant expansion along with a potential effector program for high IFN-γ production.

Abstract B130: Elevated cancer-intrinsic type I interferon signaling confers a dependency on the RNA editor ADAR1

Abstract The deamination of adenosine to inosine (known as A-to-I editing) is one of the most prevalent RNA modifications that occurs in metazoans and is mediated by the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes. The enzyme ADAR/ADAR1 catalyzes the majority of A-to-I editing where it has been demonstrated to effect coding sequence, miRNA function and silencing of Alu repetitive elements1. A critical function of ADAR1 is to edit double stranded RNA (dsRNA) structures that can activate the cytoplasmic nucleoside sensors MDA5 and PKR, preventing aberrant activation of an innate immune type I interferon (IFN) response2,3. Consistent with this hypothesis, mutations in ADAR1 and other enzymes involved in nucleoside metabolism and/or sensing are found in Aicardi-Goutiéres Syndrome (AGS), an interferonopathy associated with spontaneous interferon production4. Through analysis of pooled CRISPR and shRNA screening data, we have identified that cancer cell lines with elevated expression of specific type I interferon stimulated genes (ISGs) are dependent on ADAR1 for their survival. Tumor cells can display elevated intrinsic type I interferon signaling and associated dsRNA burden due to multiple factors, including chronic cytoplasmic DNA activation of STING, oncovirus infection and other proinflammatory signals. We have identified a core subset of 26 ISGs that predict a cellular dependency on ADAR1 and demonstrate elevated expression of these ISGs in a diverse range of tumor types including head and neck squamous cell carcinoma, breast cancer, esophageal cancer, and lung cancer. Utilizing an isogenic cell line with low basal type I interferon signaling and knockout of ADAR1, we demonstrate that we can induce cell death through exogenous treatment with the dsRNA mimetic poly-(I:C) or type I interferons (IFNα or IFNβ) but not the type II interferon IFNγ. We believe that chronic ISG expression and dsRNA burden creates a dependency on ADAR1 to prevent activation of MDA5 and PKR. These data suggest an ADAR1 inhibitor could be beneficial for the treatment of tumors with elevated ISG expression. Eisenberg et al., Nature Review Genetics (2018) Ahmad et al., Cell (2018) Chung et al., Cell (2018) Crow et al., Nature Review Immunology (2015) Citation Format: Alexandra K. Gardino, Cindy Collins, Maureen S. Lynes, P. Ann Boriack-Sjodin, Robert A. Copeland, Scott A. Ribich. Elevated cancer-intrinsic type I interferon signaling confers a dependency on the RNA editor ADAR1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B130. doi:10.1158/1535-7163.TARG-19-B130

Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown

Type I interferon (IFN) is produced when host sensors detect foreign nucleic acids, but how sensors differentiate self from nonself nucleic acids, such asdouble-stranded RNA (dsRNA), is incompletely understood. Mutations in ADAR1, an adenosine-to-inosine editing enzyme of dsRNA, cause Aicardi-Goutières syndrome, an autoinflammatory disorder associated with spontaneous interferon production and neurologic sequelae. We generated ADAR1 knockout human cells to explore ADAR1 substrates and function. ADAR1 primarily edited Alu elements in RNA polymerase II (pol II)-transcribed mRNAs, but not putative pol III-transcribed Alus. During the IFN response, ADAR1 blocked translational shutdown by inhibiting hyperactivation of PKR, a dsRNA sensor. ADAR1 dsRNA binding and catalytic activities were required to fully prevent endogenous RNA from activating PKR. Remarkably, ADAR1 knockout neuronal progenitor cells exhibited MDA5 (dsRNA sensor)-dependent spontaneous interferon production, PKR activation, and cell death. Thus, human ADAR1 regulates sensing of self versus nonself RNA, allowing pathogen detection while avoiding autoinflammation.

Adjunctive use of interferon γ inducer for treatment of patients with schizophrenia

The present paper is devoted to evaluation of clinical and immunomodulatory effect of ultra-high dilutions of antibodies to human interferon γ, included in the complex therapy of patients with schizophrenia. Materials and methods The study was carried out at the Mental Health Research Institute, Tomsk, Russian Federation. This double-blind, placebo-controlled randomised in parallel-group study enrolled 40 patients. As a part of complex therapy, patients from the main group (n=20) received anaferon, a drug containing ultra-high dilutions of affinity-purified antibodies to human interferon γ as the active pharmaceutical ingredient; patients from the comparative group (n=20) received placebo. Duration of the therapy was 30±5 days. Assessment of severity of symptoms and changes in them were made using clinical scales: Positive and Negative Syndrome Scale, Clinical Global Impression, Abnormal Involuntary Movements Scale. Spontaneous and phytohemagglutinin-induced production of interferon γ by immunocompetent cells in supernatants of 48 h whole blood culture of patients was measured by enzyme-linked immunosorbent assay (ELISA) method. The reduction of interferon-producing potential by immunocompetent cells in comparison with reference normal value was shown in total group of patients (n=40) before combined therapy. During the treatment, increase of spontaneous interferon γ production and favourable changes in psychopathological symptoms as compared with placebo were shown in subjects receiving anaferon. It was found that favourable changes in clinical symptoms assessed using clinical scales with a high degree of confidence correlated with high level of spontaneous interferon γ production. Anaferon as a part of complex therapy of patients with schizophrenia contributes to enhancement of its efficacy acting via mechanism of psychoimmunomodulation.

HTLV-1 infection interferes with immune responses to Mycobacterium tuberculosis antigens

Tuberculosis (TB) is still a major health problem. IFN-γ and TNF are critical cytokines in the control of Mycobacterium tuberculosis (Mtb) infection. It has already been demonstrated that human T cell lymphotropic virus type 1 (HTLV-1) infection leads to a spontaneous IFN- γ and TNF production, however HTLV-1 infected subjects possess a 2-4 fold increased risk of developing tuberculosis. Nevertheless, the immune mechanisms involved in this phenomenon are still unclear. The aim of this study was to evaluate immunological features of the association between HTLV-1/Mtb in order to better understand the events leading to higher susceptibility to TB observed in HTLV-1 infected subjects. This was a cross-sectional study evaluating four groups: healthy control (HC group), HTLV-1 infected subjects without TB (HTLV-1 group) or with TB (HTLV-1 + TB group), and individuals with only TB (TB group). TNF, IL-1β, and IL-17 levels were measured in supernatants of non-stimulated or PPD stimulated peripheral blood mononuclear cells (PBMCs) by ELISA. PBMCs from HTLV-1 infected individuals had a decrease (p<0.05) in TNF production following PPD stimulation (median 114.5 pg/ml, IQ range 47-189 pg/ml) compared to non-stimulated cells (202 pg/ml, IQ range 100-250 pg/ml). When we normalized the data to non-stimulated cells, IL-1β production following PPD stimulation by individuals in the HTLV-1 + TB group (3 pg/ml, IQ range 0-43 pg/ml) was lower (p<0.05) than in patients with TB (87 pg/ml, IQ range 14-151 pg/ml). Similarly, HTLV-1/Mtb co-infected individuals had lower (p<0.05) production of IL-17 (9 pg/ml, IQ range 0-41 pg/ml) when compared to TB patients (43 pg/ml, IQ range 13-150 pg/ml). Impairment in TNF, IL-1β, and IL-17 production upon stimulation with mycobacterial antigens may contribute to the higher susceptibility to Mtb infection observed in HTLV-1 infected subjects. Financial support: CNPq, CAPES.

Effect of interferon on the direction of polarization of the immune response to influenza in children

In 1900 children of different ages and 690 adults with laboratory confirmed influenza in different epidemic seasons studied levels of interferons and interleukins 4 and 10 in the serum calculating the ratio of interleukin 4, 10 to interferon gamma. There are three type of immune response to influenza depending on the clinical course. It was shown that in flu with moderate intoxication in 66.3 % of cases in children and 72.0 % of adults marked polarization on Th1 type with increase level in serum and spontaneous interferon gamma in all age groups, in which the ratio of IL-4/IFN-g from 0.8 to 2, while in severe intoxication only 33.5 and 43.9 %, respectively. In children with bronchitis immune response Th2 type and mixed Th1/Th2 type were observed in 54.6 and 33.3 % of cases respectively, and only 12.1 % of Th1 type. With influenza, pneumonia is a complication, in 76 % of cases were determined humoral immune response by Th2 type when the ratio of IL-4/IFN-g and IL-10/ IFN-g is greater than 3, due to the increase of the content of interleukin 4 and 10, while significantly reducing levels of interferon gamma. In 23.7 % of cases observed Th1/Th2 mixed type of immune response with a ratio of 2 to 3. The obtained data allow us to determine the type of immune response to influenza infection and to predict the severity of the disease and the development of complications in children and adults, and also to determine the necessity of including in the therapy drugs of immunocorrection.

Life Devoted to Viruses and Interferon: Charles Chany (1920–2013)

Life Devoted to Viruses and Interferon: Charles Chany (1920–2013)

10 CYCLOPHILIN INHIBITORS POTENTIATE INTERFERON SIGNALING THROUGH DIMINISHED PKR PHOSPHORYLATION IN HCV-INFECTED CELLS

therapy consisting of pegylated interferon alfa2b plus ribavirin; 45% achieved a SVR. Genotyping of the TLR9 SNP rs187084 and the Interleukin 28B (IL28B) SNP rs12979860 were performed via realtime PCR and melting curve analysis. Results: The overall genotype distribution of the SNP TLR9 rs187084 was 34% TT, 47% CT and 19% CC. Significant differences in the distribution of the SNP were observed between chronically infected patients and those who spontaneously cleared the HCV infection. The homozygous TT genotype was associated with chronic infection (27% spontaneous vs. 37% chronic; OR=1.38 [1.03–1.85] p =0.025). Furthermore, the homozygous CC genotype of TLR9 rs187084 was more common in patients achieving SVR (54% CC vs. 43% CC/CT). The combination with IL28B SNPs rs12979860 provided increased rates of spontaneous viral clearance (rs12979860CC/rs187084CT/CC vs. rs12979860CT/TT/rs187084TT: 53% vs. 19%, p = 5.5x10) and of treatment response (rs12979860CC/rs187084CC vs. rs12979860CT/TT/rs187084CT/TT: 75% SVR vs. 36% SVR, p = 0.003). Conclusion: The polymorphism rs187084 in the toll-like receptor 9 (TLR9) gene is associated with the outcome of HCV infection. Carriage of the TT homozygous genotype promotes the development of a chronic HCV infection and treatment failure. Additional determination of IL28B rs12979860 might improve the prediction of spontaneous viral eradication and interferon responsiveness.

Type I IFNs signaling and apoptosis resistance in glioblastoma cells

Deletion of type I IFN genes and resistance to apoptosis induced by type I IFNs are common in glioblastoma. Here we have investigated the importance of the constitutive weak IFN-signaling in the apoptotic response to IFN-α in glioblastoma cells. U87MG cells hold a deletion of type I IFN genes, whereas in T98G cells the spontaneous IFN signaling is intact. In response to IFN-α U87MG cells produce much less TRAIL, while other IFN-inducible genes were efficiently up-regulated. Alterations in TRAIL promoter sequence and activity were not observed. DNA methylation can influence TRAIL transcription but without overt differences between the two cell lines. We also discovered that TRAIL mRNA stability is influenced by IFN-α, but again no differences can be appreciated between the two cell lines. By silencing IFNAR1 we provide evidences that the spontaneous IFN signaling loop is required to sustain elevated levels of TRAIL expression, possibly through the regulation of IRF-1. Despite the presence/absence of the constitutive IFN signaling, both cell lines were resistant to IFN-α induced apoptosis. Targeting the deisgylase USP18 can overcome resistance to IFN-induced apoptosis only in T98G cells. Alterations in elements of the extrinsic apoptotic pathway, such as Bid and c-FLIP contribute to apoptotic resistance of U87MG cells. Down-regulation of USP18 expression together with the induction of ER-stress efficiently restored apoptosis in U87MG cells. Finally, we demonstrated that the BH3-only protein Noxa provides an important contribution in the apoptotic response to ER-stress in USP18 silenced cells.

Transforming growth factor-beta1 suppresses atopic dermatitis-like skin lesions in NC/Nga mice.

Atopic dermatitis is a chronic, relapsing inflammatory disorder characterized by pruritic and eczematous skin lesions. Transforming growth factor (TGF)-beta1 has been implicated in the suppression of inflammatory responses. The purpose of this study is to determine whether TGF-beta1 suppresses skin lesions in a mouse model of atopic dermatitis. We used the NC/Nga strain of mice as an in vivo model of atopic dermatitis. The effects of exogenous TGF-beta1 on atopic dermatitis-like skin lesions in NC/Nga mice were evaluated clinically, histologically and immunologically. Subcutaneous injection of recombinant TGF-beta1 macroscopically suppressed eczematous skin lesions in NC/Nga mice associated with reduced serum immunoglobulin E (IgE) levels. Histological analysis showed that TGF-beta1 significantly inhibited the infiltration of inflammatory cells such as mast cells and eosinophils into the skin of NC/Nga mice. Spontaneous interferon (IFN)-gamma production from splenocytes of NC/Nga mice was down-regulated by the treatment with TGF-beta1 and neutralizing antibody against IFN-gamma inhibited skin lesions in NC/Nga mice. The inhibitory effect of TGF-beta1 on the skin lesions lasted at least 1 week after cessation of the treatment. These findings indicate that TGF-beta1 suppressed atopic dermatitis-like skin lesions in NC/Nga mice at least in part through down-regulation of IFN-gamma. These results suggest that TGF-beta1 may have a therapeutic potential for atopic dermatitis.

Antiviral nonspecific immunity of human placenta at term: possible role of endogenous tumor necrosis factors and interferons.

The antiviral immunity of human placenta and amniotic membrane in an organ culture (OC) system was studied. Freshly isolated explants of most of the placentas at term and the amniotic membranes were found to be relatively resistant to herpes simplex virus type 1 (HSV-1), encephalomyocarditis virus (EMCV), and vesicular stomatitis virus (VSV) infections. After in vitro aging, however, the OC acquired the sensitivity to the viruses. In about 66%-90% of placentas, resistance of freshly isolated explants to the infection was observed. This indicates that the placentas displayed a constitutive immunity against the viruses. To study the role of endogenous cytokines in antiviral immunity, we added specific antibodies neutralizing IFN and TNF activities to VSV-infected OC and checked their influence on viral replication. Increases of 10-fold to 100-fold of VSV replication in the OC treated with anti-TNF-alpha, anti-IFN-alpha, anti-IFN-gamma or anti-IFN-beta sera were observed. The results indicate the importance of the endogenous cytokines in placental and amniotic membrane immunity. However, we did not observe a simple correlation between the spontaneous IFN and TNF production and the level of resistance against viruses. In view of the results, the participation of TNF and IFN in the constitutively expressed immunity of human placenta is of a more complex nature.

Selective Alteration of the Turnover of Interferon β mRNA in Peritoneal Macrophages from LPS-Hyporesponsive Mice and Its Role in the Defective Expression of Spontaneous Interferon

Basal levels of interferon (IFN)-β mRNA transcription were detected in both freshly explanted LPS-responsive (Lpsn) and LPS-hyporesponsive (Lpsd) peritoneal macrophages (PM). In vitro cultivation of PM resulted in a time-dependent reduction in the level of IFN-β mRNA, which was far more rapid in Lpsd than in Lpsn PM. Treatment of Lpsn PM with cycloheximide (CHX) resulted in a marked accumulation of IFN-β mRNA, which was not associated with an increase in IFN-β gene transcription. However, treatment of Lpsd PM with CHX did not induce accumulation of IFN-β mRNA. CHX induced the accumulation of IFN-α-4 mRNA in both Lpsn and Lpsd PM. In Lpsn PM, CHX enhanced the accumulation of two cytoplasmic factors interacting with AU-rich sequences within the 3′ untranslated region of IFN-β mRNA. We conclude that Lpsd PM exhibit an impaired capacity to stabilize IFN-β mRNA that may account for their low expression of IFN-β.

Analysis of the time course of IFN-gamma mRNA and protein production during primary murine listeriosis. The immune phase of bacterial elimination is not temporally linked to IFN production in vivo.

IFN-gamma clearly plays an important role in the murine host response against Listeria monocytogenes, but the time course of its production and its precise role in immunity remain controversial. To address these issues, we sequentially monitored IFN production and bacterial accumulation in vivo in C57B1/6 mice during primary listeriosis. IFN-gamma mRNA levels (measured by Northern blot analysis of freshly isolated splenic RNA) and serum IFN (measured by ELISA) were both maximal on day 1 of infection, decreasing steadily after day 2 to barely detectable levels by days 4 to 6. Significantly, there was no direct relationship between IFN levels and listericidal activity in vivo. Between days 1 and 3, the period of maximal IFN production, host bacterial load (assessed by quantitating live L. monocytogenes/spleen) increased approximately 10- to 50-fold. On the other hand, during the immune phase of infection (between days 5 and 7), a period when both IFN mRNA and protein were barely detectable, the host bacterial load decreased 1,000- to 10,000-fold. The paucity of IFN production in vivo during the immune phase was unexpected in light of previous reports demonstrating abundant in vitro lymphokine release by splenocytes isolated during the same time period. By direct comparisons of IFN production in vivo and in vitro, however, we could show that the late (days 6-7) peak of IFN release observed in Ag-stimulated cultures was an in vitro artifact. By contrast the pattern of spontaneous IFN release (obtained when freshly isolated cells were incubated in the absence of Ag) conformed more closely to that observed in vivo. Because listerial Ag stimulated vigorous lymphokine release in vitro, we sought to determine whether an analogous effect could be observed in vivo. In fact, even the infusion of very large doses of live bacteria (5-20,000,000/mouse) did not stimulate endogenous IFN-gamma production in mice infected for 6 to 7 days. These studies suggest three major conclusions: 1) IFN production in vivo occurs primarily during the early phase of listeriosis; 2) the dramatic decrease in bacterial numbers observed late in infection cannot be directly attributed to increased IFN production by LM-immune T cells; 3) although Ag-driven cultures of freshly isolated cells can provide useful information about the potential lymphokine-producing capabilities of Ag-specific T cells, these results have limited relevance in understanding patterns of T cell lymphokine production in vivo.

Spontaneous release of interferon gamma by intestinal lamina propria lymphocytes in Crohn's disease. Kinetics of in vitro response to interferon gamma inducers.

The spontaneous induced release of interferon gamma (IFN gamma) by cultured intestinal lamina propria lymphocytes was investigated in patients with Crohn's disease. In contrast to normal lymphocytes, intestinal lymphocytes from these patients spontaneously released IFN gamma and seemed to contain IFN gamma in their cytoplasm. Autologous peripheral lymphocytes did not release IFN gamma. When stimulated with interferon inducers lamina propria lymphocytes from Crohn's disease tissue showed an increase in IFN gamma release 24 hours after induction with no appreciable further increase over the next two days of culture, while in control cells, either peripheral or intestinal, IFN gamma release progressively increased, peaking 72 hours after induction. These findings indicate that in Crohn's disease the intestinal lymphocytes are stimulated in vivo to produce IFN gamma and that the spontaneous IFN gamma production is compartmentalised to the gut lymphocytes. These data support the concept that locally released IFN gamma has a crucial role in cell interactions in the lamina propria and contribute to the locally occurring immune phenomena in Crohn's disease, including the increased epithelial expression of major histocompatibility complex class II antigens.

Spontaneous interferon-α antibodies in a patient with pure red cell aplasia and recurrent cutaneous carcinomas

High-titered spontaneous interferon (IFN) antibodies were detected in a patient with pure red cell aplasia (PRCA), a benign mediastinal tumor, and recurrent cutaneous carcinomas. The circulating IFN antibodies reacted broadly with various human IFN-alpha subtypes (20-140 x 10(3) neutralizing units/ml serum) but not with IFN-beta or IFN-gamma, and they neutralized the antiviral activity of the patient's endogenous IFN-alpha. The IFN-alpha-binding activity was restricted to the IgG1 subclass in a nonmonoclonal manner. Whereas the PRCA repeatedly responded to immunosuppression with high-dose cyclosporin A (CSA) and CSA plus plasmapheresis, IFN antibody production continued during treatment with cyclophosphamide and CSA. Serological analysis revealed past infection with parvovirus B19 and persistent B19 IgM titers. Antibody-mediated impairment of the IFN-alpha system might have favored the development of both PRCA and the various cutaneous carcinomas in this patient.

Spontane Interferon-Freisetzung als prädiktiver Parameter für den klinischen Verlauf bei HIV-positiven Personen

In order to establish a correlation with disease progression we prospectively evaluated ten clinical and immunologic parameters in 102 consecutive HIV-positive subjects. The eight immunologic variables were: in vitro spontaneous interferon release by peripheral blood monocytic cells, alpha- and gamma-interferon production induced by Newcastle Disease Virus and PHA, Multitest Mérieux score, PHA- and CON-A-induced lymphocyte transformation, absolute number of CD4+ cells and CD4/CD8 ratio, respectively. The two baseline clinical variables were risk factor and disease presentation. Generalized Wilcoxon analysis indicated a significant correlation of one clinical (disease presentation at entry) and three immunologic variables (spontaneous interferon release, CD4+ cell number, Multitest Mérieux) with disease progression. Baseline spontaneous release of alpha, acid-labile interferon showed the best correlation with disease progression, and appeared to be significantly associated with CD4+ cell loss. Spontaneous release of acid-labile alpha interferon by mononuclear cells in vitro could be highly predictive of disease evolution in HIV-Ab positive, AIDS-free subjects.

IFN-beta production by macrophages obtained from mice undergoing graft vs host disease.

We have previously shown that when splenocytes are obtained from mice undergoing graft vs host disease (GVHD), and then placed in culture, IFN-beta is produced spontaneously in supernatants without any stimulus. Thus, when B10.D2 spleen cells are injected into sublethally irradiated BALB/c recipient mice, in vitro splenic IFN-beta production is readily apparent between days 10 to 20 post-transplantation. In the present study, experiments were carried out to characterize the cell population(s) responsible for spontaneous IFN-beta production from GVHD spleen cells. Specific antibody and C lysis of selected cell types, including T cells, B cells, and NK cells, failed to abrogate in vitro IFN production. However, IFN-producing cells from GVHD splenocytes were nylon wool adherent and could be isolated using discontinuous Percoll gradient centrifugation from the upper most large cell fractions. IFN production was also partially resistant to irradiation. Using indirect immunofluorescence and flow cytometry, IFN production was shown to be associated with MAC-1 positive cells. MAC-1 negative splenocytes demonstrated no spontaneous IFN production. Treatment of GVHD spleen cells with silica, a selective toxin for phagocytic cells, resulted in a complete inhibition of IFN production. Thus, in vitro IFN production from GVHD splenocytes appeared to come from cells having a phenotype associated with macrophages. This in vitro IFN production by macrophages represents a unique aspect of GVHD which is not typically found in tissues of normal mice.

Spontaneous IFN-beta production. A common feature of natural suppressor systems.

Natural suppression has been described in several immunologic systems in which splenocytes are not only incapable of generating an immune response, but are also able to non-specifically suppress normal splenocyte reactivity. Natural suppressor cells exist in such transitory immune deficiency states as graft vs host disease, irradiation recovery, and during neonatal development. We have found that when splenocytes from each of these three systems were placed in culture, IFN was produced spontaneously without stimulus. This spontaneous IFN production was augmented by the addition of IL-2 or granulocyte-macrophage-CSF to cell cultures. However, these lymphokines induced little or no IFN from normal splenocytes. This unusual IFN production is especially interesting since all samples have been typed to be IFN-beta. Additionally, greater IFN-beta was produced by these spleen cells during active suppression of the MLR of normal spleen cells. In fact, antibody to IFN-beta was shown to partially reverse the natural suppression mediated by graft vs host disease splenocytes in the MLR. Thus, IFN-beta production correlates extremely well with natural suppressor states. As mice begin to recover normal immune responses in all three systems, IFN-beta ceases to be produced spontaneously in culture. These findings establish a previously undescribed IFN-beta-producing splenic phenotype and suggest that IFN-beta may contribute to the immunoregulation of natural suppressor systems.

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with bhv1 and pi3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.