IFN-γ Synthesis Research Articles

Interferon-gamma signaling drives epithelial TNF-alpha receptor-2 expression during colonic tissue repair.

Interferon-gamma (IFNγ) is traditionally recognized for its pro-inflammatory role during intestinal inflammation. Here, we demonstrate that IFNγ also functions as a pro-repair molecule by increasing TNFα receptor 2 (TNFR2 protein/TNFRSF1B gene) expression on intestinal epithelial cells (IEC) following injury invitro and invivo. In silico analyses identified binding sites for the IFNγ signaling transcription factor STAT1 in the promoter region of TNFRSF1B. Scratch-wounded IEC exposed to IFNγ exhibited a STAT1-dependent increase in TNFR2 expression. Insitu hybridization revealed elevated Tnfrsf1b mRNA levels in biopsy-induced colonic mucosal wounds, while intraperitoneal administration of IFNγ neutralizing antibodies following mucosal injury resulted in impaired IEC Tnfrsf1b mRNA and inhibited colonic mucosal repair. These findings challenge conventional notions that "pro-inflammatory" mediators solely exacerbate damage by highlighting latent pro-repair functions. Moreover, these results emphasize the critical importance of timing and amount in the synthesis and release of IFNγ and TNFα during the inflammatory process, as they are pivotal in restoring tissue homeostasis.

Классические и активирующие химерные антигенные рецепторы PD-1 как элемент мультитаргетного подхода в лечении гематологических и солидных новообразований

Aim. To generate anti-PD-L1 CAR-T effectors carrying extracellular domain PD-1 as antigen-recognizing site and to study their cytolytic activity as well as to functionally assess the anti-PD-L1 CAR-T effectors in vitro with a view to apply them in multi-targeted tumor therapy.
 Materials & Methods. Chimeric antigen receptor PD-1 was constructed using molecular cloning of PD-1 antigen-recognizing region (12–170 amino acids) into mammalian expression plasmid vector adding activation and co-stimulatory domains. Primary Т-lymphocytes of healthy donor peripheral blood mononuclear fraction were derived by expanding monoclonal antibody combination on surface markers CD3/CD28. Anti-PD-L1 CAR-T effectors were obtained by lentiviral transduction of primary T-lymphocyte genome of a healthy donor. Chimeric antigen receptor PD-1 expression and transduction efficiency were assessed by flow cytofluorometry. Specific cytotoxicity of the anti-PD-L1 CAR-T effectors was analyzed in vitro by means of real-time cytotoxicity assay (RTCA) with HeLa_PD-L1 target cell line co-cultivation. The level of cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A was assessed by flow cytofluorometry using Human Th1/Th2/Th17 CBA Kit (BD, USA).
 Results. The efficiency of lentiviral transduction and the proportion of the anti-PD-L1 CAR-T effectors were 42 %. The specificity of cytotoxic response of the anti-PD-L1 CAR-T effectors with a low effector/tumor ratio (1:20) was verified during HeLa_PD-L1 co-cultivation by a 1.5-fold decrease in the cell index (CI = 0.738) versus control (CI = 1.0645). The increase in synthesis of cytokines IL-2 (1000 pg/mL), IL-6 (438.5 pg/mL), TNF-α (44 pg/mL), and IFN-γ (1034 pg/mL) during HeLa_PD-L1 target cell line co-cultivation confirms the functionality of the analyzed effector cells.
 Conclusion. Anti-PD-L1 chimeric antigen receptor was constructed and tested in vitro. Anti-PD-L1 CAR-T lymphocytes specifically recognize and promote the cytolysis of tumor target cells by increased secretion of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-2. Chimeric antigen receptor PD-1 can be modified into chimeric switch receptor (CSR) by deleting CD3ζ-domain and can be used together with other CARs without predicted non-specific toxicity.

Immunological disorders in children with chronic gastritis associated with <i>Helicobacter pylori</i> and Epstein-Barr virus

According to modern concepts, chronic gastritis is a group of phenotypically similar diseases, the basis of which is the lesion of the gastric mucosa of different genesis and different regeneration potential. Of particular interest is the group of patients (children in the presented article) in whom gastric mucosa lesions are associated with the current infectious process caused by a combination of two causative factors - Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV). The data obtained as a result of the study of cellular and humoral immunity, autoimmunity and interferon system show significant disorders of immunological reactivity in children with chronic gastritis associated with H. pylori and VEB. The imbalance of T-lymphocyte subpopulations, impaired function of B-lymphocytes, dysimmunoglobulinemia and pronounced imbalance of interferon system with a significant decrease in induced synthesis of IFN-α and IFN-γ by blood leukocytes were revealed. The suppressive effect of VEB on various links of immunity was proved, which necessitates immunocorrective treatment. The results of the study may indicate the trigger role of VEB in the development of autoimmune gastritis.

Inhibition of adult hippocampal neurogenesis induced by postoperative CD8 + T-cell infiltration is associated with cognitive decline later following surgery in adult mice

BackgroundSome patients show persistent cognitive decline for weeks, months or even years after surgery, which seriously affects their long-term prognosis and quality of life. However, most previous basic studies have focused mainly on the mechanisms of early postoperative cognitive decline, whereas cognitive decline in the longer term after surgery is less well-understood. The subgranular zone of the dentate gyrus exhibits life-long neurogenesis, supporting hippocampus-dependent learning and memory.Main textThe aim of this study was to investigate whether adult hippocampal neurogenesis (AHN) involves in cognitive decline later following surgery and to further explore the roles of CD8 + T lymphocytes infiltrating the hippocampal parenchyma after surgery in this pathological process. Cognitive function was examined in adult mice that underwent laparotomy combined with partial hepatectomy, and the results showed that cognitive decline persisted in mice who underwent surgery during the first postoperative month, even though there was a trend toward continuous improvement over time. Significantly decreased numbers of DCX + cells, BrdU + cells, and BrdU + /DCX + cells were observed on day 8 after surgery, and a significantly decreased number of NeuN + /BrdU + cells was observed on day 28 after surgery, which indicated inhibition of AHN. After surgery, T lymphocytes, the majority of which were CD8 + T cells, infiltrated the hippocampus and secreted Interferon-γ (IFN-γ). Depletion of CD8 + T cells could inhibit the increase of IFN-γ synthesis, improve hippocampal neurogenesis, and improve postoperative cognitive function. Hippocampal microinjection of IFN-γ neutralizing antibody or adeno-associated virus to knock down IFN-γ receptor 1 (IFNGR1) could also partially attenuate the inhibition of AHN and improve postoperative cognitive function.ConclusionsThese results demonstrate that postoperative infiltration of CD8 + T cells into the hippocampus and subsequent secretion of IFN-γ contribute to the inhibition of AHN and cognitive decline later following surgery.

SHP2 promotes sarcoidosis severity by inhibiting SKP2-targeted ubiquitination of TBET in CD8+ T cells.

Sarcoidosis is an interstitial lung disease (ILD) characterized by interferon-γ (IFN-γ) and T-box expressed in T cells (TBET) dysregulation. Although one-third of patients progress from granulomatous inflammation to severe lung damage, the molecular mechanisms underlying this process remain unclear. Here, we found that pharmacological inhibition of phosphorylated SH2-containing protein tyrosine phosphatase-2 (pSHP2), a facilitator of aberrant IFN-γ abundance, decreased large granuloma formation and macrophage infiltration in the lungs of mice with sarcoidosis-like disease. Positive treatment outcomes were dependent on the effective enhancement of TBET ubiquitination within CD8+ T cells. Mechanistically, we identified a posttranslational modification pathway in which the E3 F-box protein S-phase kinase-associated protein 2 (SKP2) targets TBET for ubiquitination in T cells under normal conditions. However, this pathway was disrupted by aberrant pSHP2 signaling in CD8+ T cells from patients with progressive pulmonary sarcoidosis and end-stage disease. Ex vivo inhibition of pSHP2 in CD8+ T cells from patients with end-stage sarcoidosis enhanced TBET ubiquitination and suppressed IFN-γ and collagen synthesis. Therefore, these studies provided new mechanistic insights into the SHP2-dependent posttranslational regulation of TBET and identified SHP2 inhibition as a potential therapeutic intervention against severe sarcoidosis. Furthermore, these studies also suggest that the small-molecule SHP2 inhibitor SHP099 might be used as a therapeutic measure against human diseases linked to TBET or ubiquitination.

<i>In vitro</i> antiviral activity of a double-stranded RNA sodium salt-based medicinal product against SARS-CoV-2

Scientific relevance. Innate immune activation in the early phases of COVID-19 infection and subsequent interferon induction may help control viral replication and protect cells not yet infected with SARS-CoV-2. Thus, immunostimulants that induce interferon (IFN), including double-stranded RNA-based agents, are a promising means of post-exposure prophylaxis and treatment of COVID-19 at early stages.Aim. The study evaluated the in vitro antiviral activity of a double-stranded RNA sodium salt-based medicinal product against SARS-CoV-2.Materials and methods. The authors analysed the double-stranded RNA sodium salt-based medicinal product RADAMIN®VIRO using Vero cells and the Delta variant of SARS-CoV-2 (B.1.617). The virus titre was calculated as the tissue cytopathic dose that caused 50% cell death. The authors measured the content of IFN-α and IFN-γ in the culture fluid by enzyme immunoassay and assessed the viral load by real-time polymerase chain reaction (using the cycle threshold value) and by titration (using Vero cells).Results. The studied double-stranded RNA sodium salt-based medicinal product at a concentration of 250 or 500 μg/mL induced IFN-α and IFN-γ expression by Vero cells, thus increasing their resistance to SARS-CoV-2. The authors evaluated the antiviral activity of the medicinal product based on the virus titre, viral load, and cell monolayer damage. The antiviral activity became clear 24 h after treatment, which confirmed the ability of the medicinal product to inhibit the replication of the SARS-CoV-2 virus in vitro as early as the first day after infection.Conclusions. The double-stranded RNA sodium salt-based medicinal product induced IFN-α and IFN-γ synthesis in Vero cells, increasing their resistance to SARS-CoV-2 infection in vitro. These results demonstrate the immunomodulatory and antiviral potential of the medicinal product.

Antibody-dependent enhancement of porcine reproductive and respiratory syndrome virus infection downregulates the levels of interferon-gamma/lambdas in porcine alveolar macrophages in vitro.

Fc gamma receptor-mediated antibody-dependent enhancement (ADE) can promote virus invasion of target cells, sometimes exacerbating the severity of the disease. ADE may be an enormous hurdle to developing efficacious vaccines for certain human and animal viruses. ADE of porcine reproductive and respiratory syndrome virus (PRRSV) infection has been demonstrated in vivo and in vitro. However, the effect of PRRSV-ADE infection on the natural antiviral immunity of the host cells is yet to be well investigated. Specifically, whether the ADE of PRRSV infection affects the levels of type II (interferon-gamma, IFN-γ) and III (interferon-lambdas, IFN-λs) interferons (IFNs) remains unclear. In this study, our results showed that PRRSV significantly induced the secretion of IFN-γ, IFN-λ1, IFN-λ3, and IFN-λ4 in porcine alveolar macrophages (PAMs) in early infection, and weakly inhibited the production of IFN-γ, IFN-λ1, IFN-λ3, and IFN-λ4 in PAMs in late infection. Simultaneously, PRRSV infection significantly increased the transcription of interferon-stimulated gene 15 (ISG15), ISG56, and 2', 5'-oligoadenylate synthetase 2 (OAS2) in PAMs. In addition, our results showed that PRRSV infection in PAMs via the ADE pathway not only significantly decreased the synthesis of IFN-γ, IFN-λ1, IFN-λ3, and IFN-λ4 but also significantly enhanced the generation of transforming growth factor-beta1 (TGF-β1). Our results also showed that the ADE of PRRSV infection significantly reduced the mRNAs of ISG15, ISG56, and OAS2 in PAMs. In conclusion, our studies indicated that PRRSV-ADE infection suppressed innate antiviral response by downregulating the levels of type II and III IFNs, hence facilitating viral replication in PAMs in vitro. The ADE mechanism demonstrated in the present study furthered our understanding of persistent pathogenesis following PRRSV infection mediated by antibodies.

T-Cell Immunity in COVID-19-Recovered Individuals and Individuals Vaccinated with the Combined Vector Vaccine Gam-COVID-Vac.

The COVID-19 pandemic has required extensive research on the new coronavirus SARS-CoV-2 and the creation of new highly effective vaccines. The presence of T-cells in the body that respond to virus antigens suggests adequate antiviral immunity. We investigated T-cell immunity in individuals who recovered from mild and moderate COVID-19 and in individuals vaccinated with the Gam-COVID-Vac combined vector vaccine. The ELISPOT method was used to determine the number of T-cells responding with IFN-γ synthesis to stimulation by peptides containing epitopes of the S-protein or N-, M-, ORF3, and ORF7 proteins, using peripheral blood mononuclear cells (PBMCs). At the same time, the multiplex method was used to determine the accumulation of IFN-γ and other cytokines in the culture medium. According to the data obtained, the proportion of positive conclusions about the T-cell immune response to SARS-CoV-2 antigens in control, recovered, and vaccinated individuals was 12%, 70%, and 52%, respectively. At the same time, more than half of the vaccinated individuals with a T-cell response were sensitized to the antigens of N-, M-, ORF3, and ORF7 proteins not produced by Gam-COVID-Vac, indicating a high likelihood of asymptomatic SARS-CoV-2 infection. Increased IFN-γ release by single sensitized T-cells in response to specific stimulation in recovered and vaccinated individuals did not result in the accumulation of this and other cytokines in the culture medium. These findings suggest a balance between cytokine production and utilization by immunocompetent cells as a prerequisite for providing a controlled cytokine signal and avoiding a "cytokine storm".

CYTOKINE PROFILE IN BLOOD SERUM OF INFERTILE MEN WITH CONCOMITANT PATHOLOGIES

The mechanisms of formation of male infertility are increasingly becoming immune-dependent. Immunological isolation of the testes is provided by the anatomical blood-testis barrier and the special tolerance of the immune system to antigens expressed on male gametes. For a better understanding of the immunopathogenetic mechanisms of infertility, a study of the role of various immune factors is required.
 The aim: to study the role of pro-inflammatory and anti-inflammatory cytokines in blood serum to clarify their role in spermatogenesis.
 Materials and Methods: 45 infertile men aged 22-48 were examined. They were divided into 2 groups: first group – 22 men with a systemic autoimmune disease - rheumatoid arthritis; second group – 23 somatically healthy patients with idiopathic infertility. The control group included 27 fertile healthy men aged 22-48 years. Determination of the cytokines concentration in blood serum was carried out by the immunoenzymatic method. Student's t-test was used to compare the significant difference in mean values between groups. P < 0.05 was considered significant.
 Results. Patients with autoimmune diseases (rheumatoid arthritis) had the highest rate of leukocytospermia, indicating a possible long-term inflammatory process. In patients with idiopathic infertility, oligozoospermia was diagnosed in 3 patients (13.04%), oligoasthenozoospermia in 7 patients (30.36%), asthenozoospermia in 8 patients (34.78%) and leucocytospermia in 5 patients (21.82%). In patients with rheumatoid arthritis, the concentration of IL-18 (cytokine of the IL-1 family) in the blood serum of infertile men was 1.36 times higher than that of fertile men. The level of IL-6 was 6 times higher, and the concentration of IFN-γ exceeded the norm by more than three times. The level of IL-10 was 9,4 times higher than in control group. Significant changes in the serum cytokine profile were recorded in men with idiopathic infertility. The level of pro-inflammatory cytokines increased statistically significantly: IL-18 – 1.45 times, IL-6 – 2.85 times, IFN-γ – 2.65 times. Simultaneously, the level of anti-inflammatory cytokines increased: IL-10 – 3.0 times.
 We also analyzed the ratio of serum levels of pro- and anti-inflammatory cytokines. Significant increase in ІL-10/TNF-α ratio was recorded in men with idiopathic infertility and infertile men with rheumatoid arthritis. Specifically, in men with idiopathic infertility the ІL-10/TNF-α ratio was 3.3 times higher and in patients with rheumatoid arthritis the ІL-10/TNF-α ratio was 4.1 times higher than in control group. In patients with rheumatoid arthritis the ІL-10/ІFN-γ ratio was in 3 times higher than in control group. Significant positive correlations were found in healthy fertile men: TGF-β1 – IFN-γ, IL-10 – IL-18 and IL-10 – TNF-α. In men of the control group, the synthesis of IL-18 and TNF-α was balanced by the production of IL-10. The synthesis of IFN-γ was by balanced by the production of TGF-β1.
 Conclusions. The development of infertility associated with various accompanying pathologies is related with by changes in both systemic and local immune reactivity. The pro-inflammatory cytokine profile of blood serum and a decrease in the concentration of IL-1β are observed in patients with idiopathic infertility. The largest number of deviations of immune reactivity was found in infertile men with concomitant autoimmune diseases.

Role of γδ T Lymphocytes in the Pathogenesis of Autoimmune Diseases with Skin Lesions

Autoimmune diseases are associated with a severe course, early complications, disability and early mortality. Subpopulations of γδ T cells participate in the development of autoimmune diseases, including experimental ones, contributing to tissue damage. The inflammatory functions of γδ T cells are determined by their synthesis of cytokines, including IL-17, IFNγ and TNF-α, which are usually involved in autoimmunity. Different subpopulations of γδ T cells are associated with different autoimmune diseases depending on their tissue expression, and their function may contribute to pathogenesis. In this article we review studies on the role of γδ T cells in autoimmune diseases such as rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, and scleroderma, as well as their animal models. Due to the unique properties of γδ T cells encompassing adaptive and innate immunity functions, a growing understanding of this unique T cell population sheds new light on the pathogenesis of these diseases and potentially allows new therapeutic approaches to their treatment.

The Effect of Novel Selenopolysaccharide Isolated from Lentinula edodes Mycelium on Human T Lymphocytes Activation, Proliferation, and Cytokines Synthesis.

Polysaccharides isolated from Lentinula edodes are bioactive compounds with immunomodulatory properties. In our previous studies from L. edodes mycelium, we have isolated a selenium(Se)-enriched fraction (named Se-Le-30), a mixture of linear 1,4-α-glucan and linear 1,3-β- and 1,6-β-glucans. In this study, we analyzed the effects of Se-Le-30 on the activation and proliferation of human T lymphocytes stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs) and on the production of cytokines by peripheral blood mononuclear cells (PBMCs). Se-Le-30 had effects on T cell proliferation induced by Abs against CD3 and CD28. It significantly inhibited the proliferation of CD3-stimulated CD4+ and CD8+ T cells and enhanced the proliferation of CD4+ T cells stimulated with anti-CD3/CD28 Ab. Moreover, Se-Le-30 downregulated the number of CD3-stimulated CD4+CD69+ cells, CD4+CD25+ cells, as well as CD8+CD25+ cells, and upregulated the expression of CD25 marker on CD4+ and CD8+ T cells activated with anti-CD3/CD28 Abs. Furthermore, Se-Le-30 enhanced the synthesis of IFN-γ by the unstimulated and anti-CD3/CD28-stimulated PBMCs, inhibited synthesis of IL-2 and IL-4 by CD3-stimulated cells, and augmented the synthesis of IL-6 and IL-10 by unstimulated, CD3-stimulated, and CD3/CD28-stimulated PBMCs. Together, we demonstrated that Se-Le-30 exerts immunomodulatory effects on human T lymphocytes. These observations are of importance for the prospective use of Se-Le-30 in research or as a therapeutic compound.

Lymphostatin, a virulence factor of attaching and effacing Escherichia coli, inhibits proliferation and cytokine responses of human T cells in a manner associated with cell cycle arrest but not apoptosis or necrosis.

Lymphostatin is a virulence factor of enteropathogenic E. coli (EPEC) and non-O157 serogroup enterohaemorrhagic E. coli. Previous studies using whole-cell lysates of EPEC showed that lymphostatin inhibits the mitogen-activated proliferation of bulk human peripheral blood mononuclear cells (PBMCs) and the production of cytokines IL-2, IL-4, IL-5, and IFN-γ. Here, we used highly purified lymphostatin and PBMC-derived T cells to show that lymphostatin inhibits anti-CD3/anti-CD28-activated proliferation of human CD4+ and CD8+ T cells and blocks the synthesis of IL-2, IL-4, IL-10 and IFN-γ without affecting cell viability and in a manner dependent on an N-terminal DTD glycosyltransferase motif. Such inhibition was not observed with T cells activated by phorbol 12-myristate 13-acetate and ionomycin, implying that lymphostatin targets T cell receptor signaling. Analysis of the expression of CD69 indicated that lymphostatin suppresses T cell activation at an early stage and no impacts on apoptosis or necrosis were observed. Flow cytometric analysis of the DNA content of lymphostatin-treated CD4+ and CD8+ T cells showed a concentration- and DTD-dependent accumulation of the cells in the G0/G1 phase of the cell cycle, and corresponding reduction of the percentage of cells in S phase. Consistent with this, we found a marked reduction in the abundance of cyclins D3, E and A and loss of phosphorylated Rb over time in activated T cells from 8 donors treated with lymphostatin. Moreover, the cyclin-dependent kinase (cdk) inhibitor p27kip1, which inhibits progression of the cell cycle at G1 by acting on cyclin E-cdk2 or cyclin D-cdk4 complexes, was found to be accumulated in lymphostatin-treated T cells. Analysis of the abundance of phosphorylated kinases involved in signal transduction found that 30 of 39 were reduced in abundance following lymphostatin treatment of T cells from 5 donors, albeit not significantly so. Our data provide novel insights into the mode of action of lymphostatin on human T lymphocytes.

S231: GLUCOCORTICOID AND GLYCOLYSIS INHIBITORS COOPERATIVELY ABROGATE ACUTE GRAFT-VERSUS-HOST DISEASE

Background: Although glucocorticoids(GCs) are the standard first-line therapy for acute graft-versus-host disease(aGvHD), approximately 50% of aGvHD patients have no response to GCs. The role of T cell metabolism in murine aGvHD has been recently reported. Pharmacological inhibition of glycolysis by targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3) ameliorates aGvHD. However, the question of whether glycolysis is required for the function of activated T cells and whether abnormal glycolysis is involved in the occurrence of human aGvHD need to be elucidated. Moreover, the issue as to whether GCs and metabolism regulators can cooperatively suppress T cell alloreactivity and ameliorate aGvHD remains to be elucidated. Aims: The study aimed to analyse the glucose metabolism profiles of activated T cells in aGvHD patients, and to evaluate the roles of PFKFB3-stimulated glycolysis in alloantigen-activated T cells and aGvHD induction. Furthermore, the study was performed to explore the issue of whether GCs could restore activated T cells by regulating glycolysis and the effect of GCs combined with glycolysis inhibitors on activated T cells. Methods: In this prospective nested case–control study, a total of 15 aGvHD patients at diagnosis and 15 matched non-aGvHD patients. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. Subsequently, to assess whether functional T cell activation is caused by the regulation of PFKFB3, we used lentivirus transduction for the genetic regulation of PFKFB3 in vitro and in a humanized xenogeneic aGvHD model. The effect of GCs and glycolysis inhibitors on activated T cells was further explored in vitro and in a humanized murine model of aGvHD and graft versus leukaemia (GVL). In addition, to evaluate the synergistic effect of GCs, 3PO or their combination, combination index (CI) studies were performed by using the Chou-Talalay method for drug interactions. Results: Increased glycolysis, which was characterized by elevated PFKFB3, as well as higher rates of glucose consumption and lactate production in aGvHD T cells. Importantly, in vitro treatment with glycolysis inhibitor 3PO improved the activity of T cells derived from aGvHD patients through down-regulating glycolytic activity of T cells. Genetic upregulation of PFKFB3 induced T cell proliferation and differentiation into proinflammatory T cells. In a humanized mouse model, PFKFB3-overexpressing T cells aggravated aGvHD, as characterized by high aGvHD clinical scores, pathological scores and rapid lethality. Importantly, our integrated data from patient samples in vitro showed that GCs combined with 3PO decreased proinflammatory T cells and T cell proliferation, reduced glucose consumption, lactate production and PFKFB3 expression compared with the single GCs group. Notably, the average CI values of GCs and 3PO were 0.364 for the synergistic inhibition of IFN-γ synthesis and 0.475 for the synergistic inhibition of proliferation, respectively. In a humanized murine model of aGvHD and GVL demonstrated that GCs and 3PO cooperatively reduced the alloreactivity of T cells and ameliorate aGvHD without a loss of GVL effects. Summary/Conclusion: Our data indicated that glycolysis is critical for T cell activation and the induction of human aGvHD. GCs combined with glycolysis inhibitors demonstrated synergistic effects on reducing T cell alloreactivity and ameliorating aGvHD by regulating T cell glycolysis in vitro and in vivo. Thus, GCs combined with glycolysis inhibitors promise to be a novel therapeutic strategy for aGvHD patients.

188-OR: Metabolic Blockade of Glycolysis Can Delay the Adoptive Transfer of Type 1 Diabetes

Type 1 diabetes (T1D) is a result of insulin-secreting pancreatic β-cell destruction by autoreactive CD4 and CD8 T cells. There is no cure for T1D, but determining the role of immunometabolism in promoting autoreactive CD4+ T cell effector responses may provide a novel target in delaying T1D. Systemically inhibiting glycolysis with 2-deoxyglucose (2-DG) can decrease autoreactive CD8 T cell islet infiltration. However, the effects of 2-DG on CD4 T cell diabetogenicity is unknown. Proinflammatory Th1 cells mediate β-cell destruction prefer glycolysis for maintaining effector responses, while immunosuppressive Treg cells prefer oxidative phosphorylation. We hypothesize that metabolic inhibition of glycolysis will diminish proinflammatory Th1 cell responses and delay β-cell destruction in T1D. Utilizing 2-DG, we performed an adoptive transfer of diabetogenic CD4+ T cells from NOD.BDC-2.5 or NOD.BDC-6.9 mice into NOD.scid mice. Recipient mice were systemically treated with 30 mM 2-DG in the drinking water beginning 4 days prior to adoptive transfer and monitored for hyperglycemia for 80 days post-transfer. NOD.scid mice treated with 2-DG had a significant delay in diabetes onset when transferred with either BDC-2.5 (n=14, p=0.0284) or BDC-6.9 (n=17, p=0.0249) CD4+ T cells compared to vehicle-treated groups. To define the effects of 2-DG on autoreactive CD4 T cell effector responses, NOD.BDC-6.9 splenocytes were in vitro stimulated with their cognate hybrid insulin peptide (HIP; insulin C-peptide and islet amyloid polypeptide) with or without 2-DG. Flow cytometry analysis revealed a decrease in T cell proliferation, T cell activation, and Th1 lineage commitment as shown by Ki67, CD25, and T-bet expression, respectively. Effector responses were also dampened as IFN-γ synthesis was decreased in our 2-DG treatment group compared to HIP only controls. Future studies will investigate the effects of 2-DG on CD4 T cell chemokine secretion, trafficking into the islet, and cellular metabolic profiles. Disclosure M.D.Chavez: None. R.Mcdowell: None. H.M.Tse: None.

Interferons-α and -γ in clinical veterinary practice in the prevention and treatment of infectious diseases in cattle and pigs (review)

The review article analyzes the existing world experience in the use of interferons IFN-α and -γ as well as drugs based on them in clinical veterinary practice in cattle and pigs. The selection of literary sources was carried out on the basis of their relevance and the depth of the research (search retrospectiveness is 30 years). It has been established that IFNs are widely used in the prevention and treatment of both infectious and non-infectious diseases, which can be divided into 3 groups: viral infections, oncological diseases, bacterial and aseptic diseases. The use of IFNs is due to their antiviral activity and immunoregulatory effect. Thus, IFN-α exhibits antiviral activity and is the first line of nonspecific immune defense, an inducer of IFN-γ synthesis and the main coordinator of the body's successful response to a viral infection. At the same time, IFN-γ provides immunoregulatory, anti-inflammatory and mediated antibacterial action by stimulating the production of macrophages and participating in the differentiation of lymphocytes. Clinical efficacy is expressed in a decrease or cessation of the infectious process, a decrease in leukocytosis and neutrophilia, relief of acidotic syndrome, an increase in the body weight of animals and an improvement in the general clinical condition. Currently, IFN therapy is one of the most promising and constantly expanding areas of immunopharmacology and treatment of common respiratory, gastrointestinal, obstetric-gynecological diseases in farm animals.

Reparative and Immunomodulatory Potential of Low-Molecular-Weight Fractions of Secondary Metabolites of Bacillus sp.

The experiment was performed on male BALB/c mice with modeled skin wound. Two chromatographic fractions of secondary metabolites of permafrost bacteria Bacillus sp., that represent a mixture of polyethylene-polypropylene glycols oligomers with a molecular weight from 900 to 1350 Da were used as therapeutic agents. Application of the test substances on the wound surface and their parenteral administration significantly accelerated wound epithelialization in comparison with untreated control, activated metabolic processes, and improved humoral immunity. In in vitro experiments, the fractions activated the synthesis of IFNγ and IL-4 by human peripheral blood mononuclear cells. We conclude that polyethylene-polypropylene glycol oligomers can be a part of effector molecules that determine the repair and immunomodulatory potential of secondary metabolites of permafrost microorganisms Bacillus sp.

Применение меглюмина акридонацетата как метод оптимизации терапии при коклюше у детей

Pertussis therapy is based on current ideas about the immunopathogenesis of this infection. IFN-γ synthesis inducers may be used as an alternative approach to optimize therapy. Objective. To evaluate the effect of meglumine acridonacetate on the interferon status of pertussis patients. Patients and methods. A total of 45 pertussis patients aged between 4 and 14 years were examined. The age structure of patients was as follows: in the study group there were 45.5 ± 10.6% of children aged 4–6 years and 54.5 ± 10.6% of children aged 7–14 years, in the comparison group – 47.4% and 52.6%, respectively. Results. The effectiveness of meglumine acridonacetate was studied mainly among children with mixed infections, which in the study group amounted to 68.2% (15 children), and 31.8% (7 children) had pertussis monoinfections. A comparative analysis of interferon protection showed that during treatment, the proportion of patients with IFN-γ production deficiency decreased in the study group, while average IFN-γ concentrations significantly increased as compared to the baseline. Conclusion. The use of meglumine acridonacetate contributed to positive clinical and immunological dynamics, which was reflected in improved IFN-γ and IFN-α production and a shortened recovery period. Key words: interferon inducers, IFN-γ, IFN-α, pertussis in children

Major Surgical Trauma Impairs the Function of Natural Killer Cells but Does Not Affect Monocyte Cytokine Synthesis.

Major traumatic and surgical injury increase the risk for infectious complications due to immune dysregulation. Upon stimulation with interleukin (IL) 12 by monocyte/macrophages, natural killer (NK) cells release interferon (IFN) γ that supports the elimination of the pathogen. In the present study, we investigated the impact of invasive spine surgery on the relationship between monocytes and NK cells upon exposure to Staphylococcus aureus. Mononuclear cells and serum were isolated from peripheral blood of patients before and up to 8 d after surgery and stimulated with inactivated S. aureus bacteria. NK cell and monocyte function were determined by flow cytometry. NK cells continuously lost their ability to produce IFN-γ during the first week after surgery independently from monocyte-derived IL-12 secretion. IFN-γ synthesis was minimal on day 8 and was associated with decreased expression of the IL-12 receptor and activation of transcription factors required for IFNG gene transcription. Addition of recombinant IL-12 could at least partially restore NK cell function. Pre-operative levels of growth/differentiation factor (GDF) 15 in the serum correlated with the extent of NK cell suppression and with hospitalization. Thus, NK cell suppression after major surgery might represent a therapeutic target to improve the immune defense against opportunistic infections.

Prophylactic dendritic cell vaccination in antitumor immune response and tumor growth in a breast cancer mouse model

Dendritic cell vaccines have demonstrated promising results for poorly immunogenic tumors, which may promote the generation of better immune responses in the tumor microenvironment. However, the vaccine has little been evaluated as a prophylactic option. Therefore, this study evaluates the influence of prophylactic dendritic cell vaccination on the antitumor immune response in the tumor microenvironment and on tumor growth, in an experimental model with breast cancer induced by 4T1. Therefore, Balb/c mice were separated into a vaccinated group and an unvaccinated group. Dendritic cell vaccine was differentiated and matured ex vivo from bone marrow. During the experimental period, the tumor volumes were checked periodically. The tumors were evaluated for immune cells (helper T lymphocytes and cytotoxic T lymphocytes), helper T cells (Th1, Th2, Th17, and Treg), TNF-α, and IFN-γ synthesis by Th1 and cytotoxic T lymphocytes. The vaccinated group had decreased tumor volume (14.0, 0-131.7) compared to the unvaccinated group (89.59, 0.1250-459.6) (p=0.0421). The Th1, Th2, Th17, Treg, cytotoxic T subtypes, including TNF-α and IFN-γ produced by Th1 and T cytotoxic, showed a significant increase in the vaccinated group, as did the balance of Th1/Th2 and Th1/Treg. The results showed that prophylactic vaccination with dendritic cells showed a considerable antitumor effect in the studied model by promoting an increase in the activation of important cells in the immune response and a reduction in tumor volume. The data provide evidence for timely activation of immune surveillance in the absence of tumor burden.

Involvement of TIGIT in Natural Killer Cell Exhaustion and Immune Escape in Patients and Mouse Model With Liver Echinococcus multilocularis Infection.

Alveolar echinococcosis (AE) is a lethal helminthic liver disease caused by persistent infection with Echinococcus multilocularis. Although more attention has been paid to the immunotolerance of T cells caused by E. multilocularis infection, the role of natural killer (NK) cell, a critical player in liver immunity, is seldom studied. Here, we observed that NK cells from the blood and closed liver tissue (CLT) of AE patients expressed a higher level of inhibitory receptor TIGIT and were functionally exhausted with a lower expression of granzyme B, perforin, interferon-gamma (IFN-γ), and TNF-α. Addition of anti-TIGIT (T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) monoclonal antibody into AE patients' peripheral blood mononuclear cell culture significantly enhanced the synthesis of IFN-γ and TNF-α by NK cells, indicating the reversion of exhausted NK cells by TIGIT blockade. In the mouse model of E. multilocularis infection, liver and splenic TIGIT+ NK cells progressively increased dependent of infection dosage and timing and were less activated and less degranulated with lower cytokine secretion. Furthermore, TIGIT deficiency or blockade in vivo inhibited liver metacestode growth, reduced liver injury, and increased the level of IFN-γ produced by liver NK cells. Interestingly, NK cells from mice with persistent chronic infection expressed a higher level of TIGIT compared to self-healing mice. To look further into the mechanisms, more regulatory CD56bright and murine CD49a+ NK cells with higher TIGIT expression existed in livers of AE patients and mice infected with E. multilocularis, respectively. They coexpressed higher surface programmed death ligand 1 and secreted more IL-10, two strong inducers to mediate the functional exhaustion of NK cells. Our results indicate that inhibitory receptor TIGIT is involved in NK cell exhaustion and immune escape from E. multilocularis infection.