Treg Lymphocytes Research Articles

Patients with Systemic Lupus Erythematosus Show Increased Levels and Defective Function of CD69+ T Regulatory Cells.

T regulatory (Treg) cells have a key role in the pathogenesis of chronic inflammatory and autoimmune diseases. A CD4+CD69+ T cell subset has been described that behaves as Treg lymphocytes, exerting an important immune suppressive effect. In this study, we analyzed the levels and function of CD4+CD69+ Treg cells in patients with systemic lupus erythematosus (SLE). Blood samples were obtained from 22 patients with SLE and 25 healthy subjects. Levels of CD4+CD69+ Treg cells were analyzed by multiparametric flow cytometry, and their function was measured by an assay of suppression of lymphocyte activation and through the inhibition of cytokine synthesis. We found an increased percent of CD4+CD25varCD69+TGF-β+IL-10+Foxp3− lymphocytes in patients with SLE compared to controls. In addition, a significant diminution in the suppressive effect of these cells on the activation of autologous T lymphocytes was observed in most patients with SLE. Accordingly, CD69+ Treg cells from SLE patients showed a defective capability to inhibit the release of IL-2, IL-6, IL-10, and IL-17A by autologous lymphocytes. Our findings suggest that while CD4+CD69+ Treg lymphocyte levels are increased in SLE patients, these cells are apparently unable to contribute to the downmodulation of the autoimmune response and the tissue damage seen in this condition.

AGE-DEPENDENT CHANGES OF T-REGULATORY AND Th17 SUBSET LEVELS IN PERIPHERAL BLOOD FROM HEALTHY HUMANS

Age-dependent development of Th17 and Treg lymphocyte subsets in healthy humans is studied insufficiently. The present study aimed to investigate quantitative characteristics of Th17 and Treg subsets in peripheral blood of healthy subjects for various age groups. 352 healthy humans (168 female and 184 male), one month to 85 years old, were subject to examination, including 79 infants in their first year of life; 34 children at aged 1 year to 2 years 11 months; 24, at 3 to 4 years 11 months; 28, at the age of 5 to 6 years 11 months; 25 children aged 7-8 years 11 month; 36 children aged 9 to 11 years 11 months; 39 adolescents aged 12 to 14 years 11 months; 26 adolescents aged 15 to 18 years; 25 young adults aged 20 to 35 years; 11 adults at 36 to 49 years old; 16 adults aged 59-70 years, and 9 elderly people over 70 years old. The study was performed with capillary blood in children under 2 years, and venous blood taken in elder persons. The basic and ‘minor’ subsets of peripheral blood lymphocytes were evaluated by flow cytometry using four-color staining of whole blood and following erythrocyte lysis. We used the following surface markers: CD3, CD4, CD8, CD25, CD127, CD161, CD45R0 for lymphocyte subsets detection. It has been shown that Treg percentage (a ratio of CD4 + CD25 hi CD127 low/neg in the CD3 + CD4 + gate) did not depend on age of the people under study, and can be approximated by a linear function. The absolute number of Tregs in childhood is progressively decreased and, after 10 years old, it reaches plateau values. This age-dependent relationship may be approximated by a logarithmic function. Evaluation of Th17 subset levels demonstrated a strong relative and absolute age-dependent growth of this cell subpopulation. Percentage and absolute numbers of Th17 lymphocyte (share of CD4 + CD161 + CD45R0 + in the CD3 + CD4 + gate), can be approximated by a square function. The age of 10-12 years seems to be critical to the immune system formation. We suppose the process of the immune system development to be completed at this age, and maturation of the immune cell populations is then observed. A decrease in both relative and absolute numbers of Treg and Th17 lymphocyte subsets was found in elderly persons (> 70 years old). Our data on peripheral blood Tregs and Th17 subsets, with respect to their percentage and absolute numbers in healthy humans, may be used as age-related reference values.

RELATIONSHIP OF THE T-REGULATORY CELLS NUMBER WITH THE CYTOTOXIC T-LYMPHOCYTES AND NKT-CELLS LEVELS IN PATIENTS WITH RENAL CANCER

The aim of the study was to investigate the features of the relation to the number and the phenotype of the cytotoxic T-lymphocytes and NKT-cell from regulatory T-cells content in the blood by patients with renal cell carcinoma. The study included patients with renal cell carcinoma (T3N0M0, clear cell type) at the age of 40-55 years before surgery. Lymphocyte immunophenotyping was performed by flow cytometry. It is found that in the peripheral blood of the patients with renal cell carcinoma accompanied by increased number of T-regulatory cells observed decrease content of the cytotoxic T-lymphocytes and increased levels of the NKT-cells. It is assumed that no change in the number of activated T-regulatory cells and cytotoxic T-lymphocyte determined migration from the blood. Increasing the amount of the NKT-cells in renal cancer is determined by the increase of activated and effector cells but at lower levels of the regulatory subpopulation. The content of the T-regulatory cells in healthy people weakly correlated with the effector subpopulations of T-lymphocytes. In patients with renal cancer the number of the activated T-regulatory cells is closely correlated with the various NKT-lymphocytes fractions. Moreover, if the mature and regulatory NKT-cells subset detected negative relations, so with the NKT-cells expressing CD28 and CD57 markers found positive correlations that characterizes the codirectional dynamics the activated of the regulatory and effector T-lymphocyte subpopulations levels in the background of tumor growth. A canonical analysis demonstrated that the highest significance kidney cancer patients have activated regulatory T-cells, cytotoxic T cells and NKT-cells. A canonical analysis demonstrated that the highest significance by renal cancer patients have activated regulatory T-cells, cytotoxic T-cells and NKT-cells.

Treatment of Severe Autoimmune Diseases with Autologous Hematopoietic Stem Cell Transplantation

Abstract Introduction. Autoimmune diseases are a family of more than 100 heterogeneous conditions that affect 5 to 8% of the world’s population. The etiology is still un-known but the disregulation of the regulatory T-lymphocytes play a central role inthe autoimmunity and the success of the long-term remission. Although conventional immunosuppression and new biological agents can provide disease control in severely affected patients, such treatments are rarely curative and alternative strategies are needed. Indeed, severe forms of systemic autoimmune diseases, such as multiple sclerosis (MS), systemic sclerosis (SSc), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), juvenile idiopathic arthritis (JIA), hematologic immune cytopenia (HIC) and Crohn’s disease are difficult to be treated. High-dose immunosuppressive therapy followed by autologous stem cells transplantation is reliable option for a successive treatment of this group of patients. Aim. To determine the safety of the procedure of autologous stem cell transplantation in patients with autoimmune diseases and concomitant malignant hematological disorders. Methods. During a period of 15 years (from September 2000 to September 2015) at the University Clinic of Hematology in Skopje we have treated 6 patients with autoimmune disease and concomitant hematological neoplasm. None of the patients was treated for primary autoimmune diseases. Two men and 4 women, with median age of 47 years were treated. Sjogren syndrome and multiple myeloma were found in 2 patients, polyartheritis nodosa and multiple myeloma in 1 patient, rheumatoid arthritis and acute myeloblastic leukemia in 1, systemic lupus erythematosus and non-Hodgkin lymphoma in 1; severe psoriasis and acute myeloblastic leukemia in 1 patient. Results. All treated patients are alive after trans-planted procedure, with transplant related mortality day +100: 0. Conclusion. Autologous stem cell transplantation is safe and recommended option for treatment ofpatients with autoimmune disease and hematologic neoplasm.

Muscle damage and inflammation during recovery from exercise.

Unaccustomed exercise consisting of eccentric (i.e., lengthening) muscle contractions often results in muscle damage characterized by ultrastructural alterations in muscle tissue, clinical signs, and symptoms (e.g., reduced muscle strength and range of motion, increased muscle soreness and swelling, efflux of myocellular proteins). The time course of recovery following exercise-induced muscle damage depends on the extent of initial muscle damage, which in turn is influenced by the intensity and duration of exercise, joint angle/muscle length, and muscle groups used during exercise. The effects of these factors on muscle strength, soreness, and swelling are well characterized. By contrast, much less is known about how they affect intramuscular inflammation and molecular aspects of muscle adaptation/remodeling. Although inflammation has historically been viewed as detrimental for recovery from exercise, it is now generally accepted that inflammatory responses, if tightly regulated, are integral to muscle repair and regeneration. Animal studies have revealed that various cell types, including neutrophils, macrophages, mast cells, eosinophils, CD8 and T-regulatory lymphocytes, fibro-adipogenic progenitors, and pericytes help to facilitate muscle tissue regeneration. However, more research is required to determine whether these cells respond to exercise-induced muscle damage. A large body of research has investigated the efficacy of physicotherapeutic, pharmacological, and nutritional interventions for reducing the signs and symptoms of exercise-induced muscle damage, with mixed results. More research is needed to examine if/how these treatments influence inflammation and muscle remodeling during recovery from exercise.

Immunometabolism of lymphocytes and its changes in experimental diabetes mellitus

Lymphocytes are sensitive to changes in metabolism. Metabolic changes, which develop in conditions of diabetes mellitus, especially hyperglycemia, can directly influence the immunometabolism of lymphocytes. The T cells express a series of glucose transporters, the main of which is the Glut 1. The prodiabetogenic Th1 and Th17-cells that cause insulitis are characterized by high level of expression of Glut 1 and tendency to glycolysis. The suppressor Treg, on the contrary, has the low expression of Glut 1 and the high rate of oxidative metabolism. Purpose of the study: to analyze the contemporary literature and own data, obtained concerning the immunometabolism of lymphocyte and its changes in conditions of diabetes. To determine the role of 6 key metabolic ways that play a crucial role in the differentiation and survival of immune cells: 1) glycolysis; 2) tricarboxylic acid (TCA) cycle; 3) pentose-phosphate cycle; 4) fatty acid oxidation; 5) fatty acid synthesis and 6) metabolism of amino acids, each of which have different activity level in specific types of immune cells. Conclusions: different types of immune cells prefer different ways of metabolism. The effector Th1-, Th2-, Th17-cells and М1-macrophages use primarily glycolysis, pentose-phosphate cycle and synthesis of fatty acids, while T-regulatory, CD8 + memory cells and M2-macrophages use the TCA cycle and oxidation of fatty acids. Changes in the metabolism of different amino acids can influence the generation of effector and Treg lymphocytes. The high activity of mTOR can enhance the progression of diabetes by activating the effector proinflammatory subpopulations of lymphocytes, and vice versa, the low activity promotes the differentiation of Treg, blocking the insulitis. In our work we investigated the level of expression of mRNA of genes Glut 1, mTOR and AMPK1α in PLN of rats with experimental streptozotocin-induced diabetes and after metformin introduction and found that the hyperglycemia caused the transcription induction of the gene of glucose transporters Glut 1 in PLN cells. The increase of the level of mRNA genes of glucose transporters Glut 1 and protein kinase mTOR in immune cells in diabetes, which we determined, is an important trigger of their differentiation into effector proinflammatory Th1 and Th17 subpopulations.

Infiltration of Regulatory T Lymphocytes Impair Clinical Outcome in Ovarian Cancer Patients

Background: Ovarian carcinoma (OC) is one of the most severe and lethal cancers in women. Regulatory T (Treg) cells may participate in mediating a suppressive microenvironment in tumor tissue and contribute to tumor evasion from immune response. Specifically, some studies have indicated that Treg cell infiltration in malignant ascites and peripheral blood has been associated with poor clinical outcome. Methods: The number of CD4+, CD25+, forkhead box p3+ (Foxp3+) lymphocytes was assessed via flow cytometry in ascites and peripheral blood from 24 women with epithelial ovarian cancer (EOC). Overall survival was associated with Treg levels. Results: The study indicated an increase in the Treg cell subset from peripheral blood of EOC patients compared with those in healthy controls. Treg infiltration in ascites was higher than in blood into EOC patients. The patients with high infiltration of Treg in blood revealed less overall survival compared to a counterpart with normal levels of Treg. Regarding the association between patients with increased levels of Treg in ascites with overall survival there was no disadvantage found, compared to ascites from patients with normal levels of Treg. Conclusion: Study suggests that Treg lymphocytes were increased in ascites and peripheral blood in EOC patients and could be associated with diminished overall survival, via impairment antitumor immune response.

Gene Networks and Canonical Pathways Analysis in Classical Hodgkin Lymphoma Patients

Introduction: Molecular alterations involved in development of classical Hodgkin lymphoma (cHL) are only partially known. Genetic alterations in NFkB pathway and the imbalance of T regulatory (Treg) and TH17 lymphocytes has been recognized as critical pathogenetic mechanism involved in immune scape and blockade of apoptosis in Reed-Sternberg cells. Emerging evidences suggests that NFkB activation can promote lymphocyte proliferation and survival, and therapeutic inhibition of the NFkB pathway have been proposed. Recently, our group showed an increase of circulating Treg cells in patients with cHL at diagnosis, however, after therapy, elevated levels of circulating TH17 cells was observed. Increased frequencies of Treg lymphocytes in the tumor microenvironment and peripheral blood have been proposed as one of the mechanisms for this anergic state. TH17 exhibit effector functions in immune system and tumor-infiltrating TH17 cells are associated with better prognosis in human.Objectives: In this study we aimed to evaluate the immune gene expression profile in whole blood of cHL patients at diagnosis and after treatment, and evaluate pathways and gene interaction networks.Methods: This is an open multicenter study and, so far, we included 51 patients consecutively from February 2011 to November 2015. Twenty consecutively diagnosed cHL patients, with whole blood RNA extracted at diagnosis and after treatment, were recruited for this study and prospectively evaluated. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. The general expression of 96 messengers RNAs present in the peripheral blood and involved in immune response was performed by a customized quantitative real-time PCR array (TaqMan¨ Low Density Array). The data was normalized with B2M mRNAs levels and relative gene expression was calculated by the 2^DDCt method, considering Wilcoxon test and Benjamini-Hochberg adjustment to correct p-values. The differentially expressed genes were used to conduct functional/pathway enrichment analysis and construct gene interaction networks. Functional annotation networks were generated using Ingenuity Pathway Analysis (IPA; Ingenuity Systems) software.Results: From the 20 patients included for this study, 12 (60%) were male, 5 (31%) had Epstein Barr virus related cHL, 18 (90%) patients presented with B symptoms, 19 (95%) patients had advanced diseases at diagnosis (stage IIBX, III and IV) and 10% of patients relapsed. We considered paired samples from 15 patients before and after treatment. At diagnosis we observed that cHL patients presented higher expression of CD274 (2 fold, p=0.018), CD28 (1.5 fold, p=0.041), CTLA-4(1.5 fold, p=0.004), FAS (1.4 fold, p=0.041), ICOS (2.1 fold, p=0.015) and IL-10 (2.7 fold, p=0.002), and decreased expression of CCL2 (-2.7 fold, p=0.026), CCL5 (-1.6 fold, p=0.012), CD40 (-1.9 fold, p=0.003), CSF2 (-1.9 fold, p=0.012). We found no association between clinical and epidemiological characteristics with immune gene expression profile. We have found that after treatment, patients displayed a specific gene expression profile related to the top 5 canonical pathways summarized in following figure:We also built a biological network to investigate the connection between the differentially expressed genes and to predict the status (activation/inhibition) of the other connected genes (nodes). The in silico analysis revealed that NFkB, a node which its signaling activation is predicted to be activated before treatment and inhibited after (following figures). The network also showed different central node molecules (molecules connected with multiple other nodes from the network), that are indirectly related to Hodgkin lymphoma and should be further investigated as potential drugable targets.Conclusions: In this study, we showed that, at diagnosis, cHL patients presented an inflammatory gene expression profile in blood that changes after treatment to an effector/immunological one. NFkB is predicted to be activated before treatment and inhibited after ABVD chemotherapy and radiotherapy. This kind of system biology approach helped us to understand that cHL associated immunosuppression and the immune reconstitution after treatment maybe the key to develop new prognostic factors and treatment strategies as well as identify drugable targets. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Vitro Comparison of Different TKI Activity in T-Cell Populations: Selective Sparing of Treg By Nilotinib

IntroductionChronic Graft Versus Host Disease (cGVHD) is a major complication of allogeneic stem-cell transplantation and is characterized by frequent multi-organ involvement that resembles the autoimmune diseases. Donor-derived CD4+ and CD8+ T lymphocytes have classically been considered to be the main effector cells mediating GVHD pathogenesis. Indeed, removal of T cells from transplant inocula almost completely prevents GVHD developing, at the price of increased incidences of graft rejection and disease recurrence. However recent studies suggest that B cells might also play an important role in the biology of cGVHD. The role of Treg lymphocytes in the pathogenesis of cGVHD is still controversial and the tyrosine kinase inhibitor′s (TKI) role in the modulation of this pathway is not yet fully characterized. In vitro data confirm that TKIs regulates both innate and adaptive immune response by interacting with many cell population such as T-cells, B-cells, dendritic cells, mast cells and macrophages.According to these observations, we investigated the TKI′s immunomodulatory effects (Nilotinib, Dasatinib, Imatinib, Ponatinib) on lymphocyte populations.Materials and MethodsPeripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Biocoll. Cells were cultured in RPMI 1640 at a concentration 1x106 cell/well. Nilotinib, Imatinib, Dasatinib and Ponatinib were added to cell cultures at serial concentration (Imatinib:1μM,10μM,50μM; Nilotinib:0.5μM,2μM,10μM; Dasatinib:50nM,100nM,200nM; Ponatinib:1nM,10nM,50nM,100nM) on the first day.Six-color flow cytometry analysis (Facs Canto II) was performed on the cells harvested after 96 h cultures using conjugated antibodies (CD3,CD4,CD16,CD56,CD3,CD25,CD19,CD45RA,FoxP3,CD127,7-Aminoactinomycin-D), for cell cycle analysis cells were stained with propidium iodide.For cytokine analysis, supernatants were collected and analyzed for cytokines according to the instruction of Bio-Plex Pro Human Cytokine 17-plex Assay with Bio-Plex (Bio-Rad).ResultsA significant decrease of cytotoxic T cells viability was observed when cells were cultured in presence of Imatinib (50μM,p<0.01), Ponatinib (10nM,p<0.05) and Dasatinib (100nM,p<0.01). On the contrary, exposure to Nilotinib didn′t induce cell death. Increasing concentrations of all the tested TKI significantly inhibited T cell proliferation in a dose-dependent manner; the effect become statistically significant starting from Imatinib (1μM,p<0.05), Dasatinib (50nM,p<0.01), Ponatinib (50nM,p<0.01) and Nilotinib (0.5μM,p<0.01).Exposure to Imatinib, Dasatinib and Ponatinib induced a statistically significant decrease (p<0.01) of Treg cells proportion, even at the lowest drug concentration in culture; Nilotinib induced Treg decrease only at concentrations exceeding 2μM (p<0.01), higher than those usually achieved in clinical practice.A significant increase of naive Treg apoptosis was observed after exposure to Dasatinib (50nmM,p<0.01), Ponatinib (50nM,p<0.01) and Imatinib (50μM,p<0.01); exposure to Nilotinib has no effect on this population.Both Nilotinib and Dasatinib induced a profound inhibition of pro-inflammatory cytokine production (in particular TNFα, IFNγ, IL13 and IL17) when added to the cell cultures (p<0.05); slower decrease in supernatant cytokine concentration was observed in presence of either Imatinib (50μM,p<0.05) and Ponatinib (50nM,p<0.05).Increasing concentrations of all TKIs except Nilotinib induced a significant decline of NK cells (p<0.01) and B cell (p<0.01).ConclusionThe present study focuses the peculiar Nilotinib activity on lymphocyte′s regulation: this TKI, at therapeutic concentrations in vitro, interact with innate and adaptive immune response show anti-inflammatory properties. Unlike other TKIs, Nilotinib determine inflammatory cytokines reduction, preserving T cell population and Treg. These data support the potential use of Nilotinib in cGVHD [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantitative and functional analysis of CD69+ T regulatory lymphocytes in patients with periodontal disease.

Periodontal disease is chronic inflammatory process that affects the attachment structures of the teeth and constitutes a significant cause of tooth loss in adults. Although different bacteria play an important role in the triggering of this condition, the progression and severity of the disease are strongly affected by the host immune response, which is under the control of different immune regulatory mechanisms, including T regulatory (Treg) cells. The aim of this study was to assess the frequency and function of CD69+ Treg lymphocytes in patients with chronic periodontal disease. Peripheral blood samples (n = 33) and gingival tissue (n = 9) were obtained from patients with chronic periodontal disease. Blood samples from 25 healthy individuals were also studied. Levels of CD69+ Treg lymphocytes in peripheral blood and gingival tissue were determined by six-color multiparametric flow cytometry, immunofluorescence, and immunohistochemistry. The immune regulatory function of CD69+ Treg cells was tested by an in vitro assay of inhibition of lymphocyte activation. Percentages of CD69+ Treg cells were significantly higher in the peripheral blood from patients with active periodontal disease compared to healthy controls, and these percentages inversely correlated with the periodontal attachment loss. Increased numbers of these Treg cells were detected in the gingival tissue from active PD patients compared to their peripheral blood. However, the suppressive function of CD69+ Treg cells was significantly diminished in patients with periodontal disease compared to healthy controls. Our data suggest that CD69+ Treg cells seem to be another important piece in the complex immunopathogenesis of periodontal disease.

Mesenchymal Stem Cells Attenuate the Adverse Effects of Immunosuppressive Drugs on Distinct T Cell Subopulations.

Immunosuppressive drugs are widely used to treat undesirable immune reaction, however their clinical use is often limited by harmful side effects. The combined application of immunosuppressive agents with mesenchymal stem cells (MSCs) offers a promising alternative approach that enables the reduction of immunosuppressive agent doses and simultaneously maintains or improves the outcome of therapy. The present study aimed to determinate the effects of immunosuppressants on individual T cell subpopulations and to investigate the efficacy of MSC-based treatment combined with immunosuppressive drugs. We tested the effect of five widely used immunosuppressants with different action mechanisms: cyclosporine A, mycophenolate mofetil, rapamycin, and two glucocorticoids - prednisone and dexamethasone in combination with MSCs on mouse CD4+ and CD8+ lymphocyte viability and activation, Th17 (RORγt+), Th1 (T-bet+), Th2 (GATA-3+) and Treg (Foxp3+) cell proportion and on the production of corresponding key cytokines (IL-17, IFNγ, IL-4 and IL-10). We showed that MSCs modulate the actions of immunosuppressants and in combination with immunosuppressive drugs display distinct effect on cell activation and balance among different T lymphocytes subpopulations and exert a suppressive effect on proinflammatory T cell subsets while promoting the functions of anti-inflammatory Treg lymphocytes. The results indicated that MSC-based therapy could be a powerful strategy to attenuate the negative effects of immunosuppressive drugs on the immune system.

37P - Immunological markers of DC-based vaccine antitumor efficacy in patients with NSCLC

Aim/Background: One of the topical areas of cancer biotherapy is the development of new methods of specific active immunotherapy (IT), including dendritic cells (DC) based vaccines. The essential problem in achieving maximum therapeutic effect of antitumor vaccines is the lack of immunological criteria for evaluating their efficacy. The aim of this study was to analyze the immunological parameters in patients with non-small lung cancer (NSCLC) after DC-IT. Methods: DC were generated from monocytes of peripheral blood with the addition of growth factors (GM-CSF, IL-4) and loaded with autologous lyophilized tumor antigens. Adjuvant DC vaccine has been included in treatment of 60 patients with IIB-IIIA NSCLC stages after primary treatment. DC-vaccine was injected 4-times with intervals of 1 month. More than 25 immunological parameters that characterize the immune system of patients were analyzed with flow cytometry and real-time PCR at all stages of IT. Results: Depending on the clinical course of the disease, patients were divided into 2 subgroups: patients with disease stabilization (DS, n = 52) and with disease relapse (DR, n = 8). Before IT in DR group the number of T-reg lymphocytes significantly increased comparing to DS and was (10,21±2,44)% against (5,18±0,91)% in DS group (p < 0.05); TGF-β expression was 3 times lower than in DS (p < 0.05). Also, in DR group MIP-1α expression was 10 times higher than RANTES expression level, and during IT has increased more than 5 times. In DS group the level of chemokines expression was opposite. RANTES expression level was 5.9 times higher than MIP-1α. Increase of CD4 + cells number in DS patients was observed on the 3rd and 4th phases of DC-IT (p < 0.05) and was 2.2 times higher than in DR (p < 0.02). Conclusions: Assessment of immunological efficiency of DC-IT allowed to establish the most informative prognostic markers such as the number of T-reg cells, mRNA expression levels of TGF-β, MIP-1α, RANTES and the number of CD4 + -cells memory. Legal entity responsible for the study: National Cancer Institute of MPH of Ukraine Funding: National Cancer Institute of MPH of Ukraine Disclosure: All authors have declared no conflicts of interest.

Macrophage-mediated psoriasis can be suppressed by regulatory T lymphocytes.

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis-like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro-inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis-like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF-mediated psoriasis, ihTNFtg mice were crossbred into RAG1KO mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1KO or wild-type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild-type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3-positive cells into RAG1KO or wild-type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro-inflammatory activity of macrophages, which are the major immune effector cells in hTNF-mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pathogenesis of thyroid autoimmune disease: the role of cellular mechanisms

Hashimoto's thyroiditis (HT) and Graves’ disease (GD) are two very common organ-specific autoimmune diseases which are characterized by circulating antibodies and lymphocyte infiltration. Although humoral and cellular mechanisms have been classically considered separately in the pathogenesis of autoimmune thyroid diseases (AITD), recent research suggests a close reciprocal relationship between these two immune pathways. Several B- and T-cell activation pathways through antigen-presenting cells (APCs) and cytokine production lead to specific differentiation of T helper (Th) and T regulatory (Treg) cells. This review will focus on the cellular mechanisms involved in the pathogenesis of AITD. Specifically, it will provide reasons for discarding the traditional simplistic dichotomous view of the T helper type 1 and 2 pathways (Th1/Th2) and will focus on the role of the recently characterized T cells, Treg and Th17 lymphocytes, as well as B lymphocytes and APCs, especially dendritic cells (DCs).

Human trophoblastic β1-glycoprotein as a differentiation factor of minor regulatory T-lymphocyte subsets (Treg, Th17). The involvement of CD9

Human trophoblastic β1-glycoprotein (PSG) was studied in vitro as a differentiation factor of T-regulatory lymphocytes (Treg) and IL-17-producing lymphocytes. The role of CD9 molecules in realization of PSG effects was evaluated using anti-CD9 monoclonal antibodies. A human heterogeneous PSG was produced according to the original authors’ technique. It was revealed that PSG (10 or 100 μg/mL) increased the number of Treg (CD4+FOXP3+) and promoted the expression of CTLA-4 and GITR in these cells. It was found that PSG (10 and 100 μg/mL) impeded differentiation of the CD4+ cells into Th-17 lymphocytes (ROR-γt+IL-17A+). Some of the effects exerted by PSG (100 μg/mL) on the Treg/Th-17 differentiation was abolished upon the blockade of CD9 by antibodies; this concerned, in particular, the expression of FOXP3, CTLA-4, GITR, and ROR-γt. However, the depressing effects of PSG (100 μg/mL) on the expression and production of IL-17A did not depend on CD9. Thus, PSG favors the differentiation of CD4+ cells into Treg and suppresses the induction of Th17; some of the effects require the involvement of CD9.

Relationship Between Different Subpopulations of Circulating CD4+ T-lymphocytes and Microvascular Structural Alterations in Humans.

Different components of the immune system, including innate and adaptive immunity (T-effector lymphocytes and T-regulatory lymphocytes-TREGs) may be involved in the development of hypertension. In addition, it was demonstrated in animal models that TREGs may prevent angiotensin II-induced hypertension and vascular injury/inflammation. However, no data are presently available in humans about possible relationships between T-lymphocyte subtypes and microvascular structural alterations. For this purpose, in the present study, we enrolled 24 normotensive subjects and 12 hypertensive patients undergoing an elective surgical intervention. No sign of local or systemic inflammation was present. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance arteries were dissected and mounted on a wire myograph and the media to lumen ratio (M/L) was calculated. In addition, retinal arteriolar structure was evaluated noninvasively by scanning laser Doppler flowmetry. Capillary density in the nailfold, dorsum of the finger, and forearm were evaluated by videomicroscopy. A peripheral blood sample was obtained before surgery for assessment of T-lymphocyte subpopulations by flow cytometry. Significant negative correlations were observed between indices of microvascular structure (M/L of subcutaneous small arteries and wall to lumen ratio of retinal arterioles) and circulating TREG lymphocytes. A direct correlation was observed between M/L of subcutaneous small arteries and circulating Th17 lymphocytes. In addition, total capillary density was correlated with a TREG effector memory subpopulation. Our data suggest that some lymphocyte subpopulations may be related to microvascular remodeling, confirming previous animal data, and opening therapeutic possibilities.

Coincidence of PTPN22 c.1858CC and FCRL3 -169CC genotypes as a biomarker of preserved residual β-cell function in children with type 1 diabetes.

Genotype-phenotype studies in type 1 diabetes (T1DM) patients are needed for further development of therapy strategies. Our aims were to investigate the distribution of selected PTPN22 and FCRL3 gene polymorphisms and their associations with clinical course of disease in children with newly diagnosed T1DM from the Pomeranian region of Poland. The prospective, longitudinal study of 147 children with newly diagnosed T1DM-autoimmune subtype was conducted. The PTPN22 c.1858T>C (rs2476601) and FCRL3 -169C>T (rs7528684) polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and DNA sequencing. The frequencies of genotypes were compared between the study and population-matched control group (327 random anonymous samples from the Pomeranian region). Selected patients underwent a 24-monthly follow up [periodic re-evaluation of fasting C-peptide concentration (FCP) and hemoglobin A1c (HbA1c ) level]. A significantly lower coincidence of the PTPN22 c.1858CC and FCRL3 -169CC genotypes was found in the study group compared with controls (P = 0.04). The PTPN22 c.1858CC and FCRL3 -169CC genotype combination, restricted to female patients only, was associated with well-preserved residual β-cell function throughout the entire follow up (prolonged FCP level increase up to the sixth month of disease, with further very stable dynamics-FCP median level ≥0.67 ng/mL without significant decrease up to the 24th month). HbA1c levels in this subgroup also remained the lowest during the observation period. Ascertained phenomenon could be explained by an interacting mechanism of the two polymorphisms through estrogen-regulated nuclear factor kappa B signaling in regulatory T (Treg ) lymphocytes. This hypothesis, if confirmed, may lead to further development of Treg administration-based therapies.

OP.3D.06] RELATIONSHIP BETWEEN DIFFERENT POPULATIONS OF CIRCULATING T LYMPHOCYTES AND MICROVASCULAR OXIDATIVE STRESS

Objective: Both innate and adaptive immune systems may contribute to the pathogenesis of cardiovascular disease and vascular remodeling through inflammation and oxidative stress. Particularly, the balance between Th1 effector lymphocytes (producing interferon-γ) and T regulatory (Treg) lymphocytes, which elicit an anti-inflammatory activity, may be crucial for blood pressure elevation and organ damage development, at least in experimental models. In particular, Tregs have been previously demonstrated to inversely correlate with subcutaneous small resistance artery media to lumen ratio (M/L) and retinal arteriole wall to lumen ratio (W/L) (unpublished data). Therefore, we evaluate the possible relationship between Th1 or Treg lymphocytes and microvascular oxidative stress. Design and method: We enrolled 11 normotensive subjects and 4 hypertensive patients undergoing an election surgical intervention. No sign of local or systemic inflammation was present in any subjects or patients. All patients underwent a biopsy of subcutaneous fat during surgery. A peripheral blood sample was obtained before surgery for assessment of T lymphocyte populations. Lymphocyte phenotype was evaluated by flow cytometry after 5 hour in vitro activation in order to assess Th1 lymphocytes. Superoxide anion production, as index of oxidative stress, was evaluated in the bioptic tissue by florescent dye dihydroethidium and quantified using an image analyzer. Results: Results are summarized in the Table (RTE: recent thymus emigrant; EM: effector memory).A significant positive correlation was observed between Th1 lymphocytes and superoxide anion production in subcutaneous microvessels. Additionally, a significant inverse correlation was observed between microvascular free radical production and Total Treg, RTE and EM Tregs. Conclusions: Our data suggest that Treg lymphocytes may be protective against microvascular damage, probably because of their anti-oxidant properties confirming an involvement of adaptive immune system on microvascular damage.

PP.21.05] RELATIONSHIP BETWEEN MICROVASCULAR STRUCTURE AND T REGULATORY LYMPHOCYTES OF SMALL RESISTANCE ARTERIES

Objective: Recently it has been demonstrated a role for adaptive immunity, particularly for T regulatory lymphocytes (Tregs), in the development of hypertension and in preventing of angiotensin II–induced vascular injury and inflammation in animal models (Barhoumi T et al, Hypertension 2011;57:469–476). However, no data are presently available in human beings about possible relationships between Tregs and microvascular structural alterations. Design and method: In the present study we enrolled 11 normotensive subjects and 8 hypertensive patients undergoing an election surgical intervention. All patients underwent a biopsy of subcutaneous fat during surgery. Subcutaneous small resistance artery structure was assessed by wire myography and media to lumen ratio (M/L) was calculated. W/L of retinal arterioles was obtained by Scanning Laser Doppler Flowmetry. Functional (basal) and structural (total) microvascular density were studied by capillaroscopy before and after venous congestion. No sign of local or systemic inflammation was present in any subjects or patients. We extracted genomic DNA from small resistance arteries and analyzed methylation status of the FoxP3 gene promoter involved in Treg lymphocytes activation. Unmethylated FoxP3 has been demonstrated to be specific for Treg lymphocytes. A peripheral blood sample was obtained before surgery for routine chemistry Results: Results are summarized in the Table.A significant positive correlation was detected between Tregs in small resistance arteries and basal, total and delta gain capillary density in the forearm, whereas no correlations were observed with small resistance artery M/L and retinal arteriole W/L. In addition, a significant inverse correlation was detected between Treg in subcutaneous small vessels and C reactive protein. Conclusions: Our data suggest that Treg lymphocytes detected in subcutaneous small resistance artery wall are related with capillary density and inversely related with inflammatory markers suggesting a protecting role of Treg also, probably, in terms of angiogenetic properties.

Abstract # 1703 Coping strategies of top managers, the state of theirs immune systems and risk of cancer

We would like to introduce our project focused on relation between coping strategies and state of immune system. The project is focused on comparison between two groups of people. First group is top managers, where we expect significant long-term stressfull working conditions. Second group are people (with similar characteristics compared to the first group) who were affected by cancer in recent history. Following the comparison of results from these groups, provides a foundation for gaining interesting information both in the field of coping strategies and approaches to managing life worries, but also values of the immune system. We use combination of two metodological approaches. From the area of psychology we use several questionnaires focused on perceived level of stress, preferred coping strategies and personality. We use following testing methods: Stress Profile (Novack, 2006), Bochum Inventory of Personality (Hossip, 2011), Coping Strategy Inventory (Tobin, 2001), Overal Psychological Resistance (Miksik, 2004). From the area of immunology we use detail immunological examination (taken from blood sample) with main focus on: NK cells, T-cytotoxic lymphocytes, T-helpers lymphocytes, T-regulatory lymphocytes, proinflammatory parameters and tumor markers. We are going to present result of the first phase of the project, executed on the sample of participants.