Ex vivo characterization of Breg cells in patients with chronic Chagas disease
Ontology highlight
ABSTRACT:
SUBMITTER: Girard M
PROVIDER: S-EPMC7943772 | biostudies-literature | 2021 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The discovery of T cell epitopes is essential not only for gaining knowledge about host response to infectious disease but also for the development of immune-intervention strategies. In Chagas disease, given the size and complexity of the Trypanosoma cruzi proteome and its interaction with the host's immune system, the fine specificity of T cells has not been extensively studied yet, and this is particularly true for the CD4+ T cell compartment. The aim of the present work was to optimize a protocol for the generation of parasite-specific memory T cell lines, representative of their in vivo precursor populations and capable of responding to parasite antigens after long-term culture. Accordingly, peripheral blood mononuclear cells (PBMC) from both chronic asymptomatic and cardiac patients, and from non-infected individuals, underwent different in vitro culture and stimulation conditions. Subsequently, cells were tested for their capacity to respond against T. cruzi lysate by measuring [3H]-thymidine incorporation and interferon-? and GM-CSF secretion. Results allowed us to adjust initial T. cruzi lysate incubation time as well as the number of expansions with phytohemagglutinin (PHA) and irradiated allogeneic PBMC prior to specificity evaluation. Moreover, our data demonstrated that parasite specific T cells displayed a clear and strong activation by using T. cruzi lysate pulsed, Epstein-Barr virus (EBV)-transformed human B lymphocytes (B-LCL), as autologous antigen presenting cells. Under these culture conditions, we generated a clone from an asymptomatic patient's memory CD4+ T cells which responded against epimastigote and trypomastigote protein lysate. Our results describe a culture method for isolating T. cruzi specific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells.
| S-EPMC5446171 | biostudies-literature
Project description:A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.
| S-EPMC6345919 | biostudies-literature
Project description:Chronic Chagas disease cardiomyopathy (CCC) is the most frequent and severe form of this parasitic disease. CCC is caused by a progressive inflammation in the heart, resulting in alterations that can culminate in heart failure and death. The use of dendritic cells (DCs) appears as an option for the development of treatments due to their important role in regulating immune responses. Here, we investigated whether tolerogenic cells (tDCs) could interfere with the progression of CCC in an experimental model of Chagas disease. The tDCs were generated and characterized as CD11b+ CD11c+ cells, low expression of MHC-II, CD86, CD80, and CD40, and increased expression of PD-L. These cells produced low levels of IL-6 and IL-12p70 and higher levels of IL-10, compared to mature DCs (mDCs). Interestingly, tDCs inhibited lymphoproliferation and markedly increased the population of FoxP3+ Treg cells in vitro, compared to mature DCs. In a mouse model of CCC, treatment with tDCs reduced heart inflammation and fibrosis. Furthermore, tDCs treatment reduced the gene expression of pro-inflammatory cytokines (Ifng and Il12) and of genes related to cardiac remodeling (Col1a2 and Lgals3), while increasing the gene expression of IL-10. Finally, administration of tDCs, increased the percentage of Treg cells in the hearts and spleens of chagasic mice. Ours results show that tolerogenic dendritic cells have therapeutic potential on CCC, inhibiting disease progression.
| S-EPMC7154094 | biostudies-literature
Project description:Chagas disease, a neglected tropical disease (NTD) caused by the flagellated protozoan Trypanosoma cruzi (T. cruzi), is a major public health problem. It was initially restricted to Latin America, but it is now expanding globally. Host and pathogen interactions are crucial in the establishment of disease, and since 1970, it has been known that eukaryotic cells release extracellular vesicles (EVs), which in turn have an important role in intercellular communication in physiological and pathological conditions. Our study proposed to characterize and compare circulating EVs isolated from the plasma of chronic Chagas disease (CCD) patients and controls. For this, peripheral blood was collected from patients and controls, and mononuclear cells (PBMCs) were isolated and stimulated with parasite EVs, showing that patient cells released fewer EVs than control cells. Then, after plasma separation followed by EV total shedding enrichment, the samples were subjected to ultracentrifugation to isolate the circulating EVs, which then had their size and concentration characterized by nanoparticle tracking analysis (NTA). This showed that patients had a lower concentration of circulating EVs while there were no differences in size, corroborating the in vitro data. Additionally, circulating EVs were incubated with THP-1 cells (macrophages) that, after the interaction, had their supernatant analyzed by ELISA for cytokine detection. In relation to their ability to induce cytokine production, the CCD patient EVs were able to induce a differential production of IFN-γ and IL-17 in relation to controls, with differences being more evident in earlier/less severe stages of the disease. In summary, a decreased concentration of circulating EVs associated with differential activation of the immunological system in patients with CCD is related to parasite persistence and the establishment of chronic disease. It is also a potential biomarker for monitoring disease progression.
| S-EPMC7815414 | biostudies-literature
Project description:Natural Killer T (NKT) cells have gained widespread attention among immunologists because of their distinct ability to regulate anti-tumor responses and to influence the outcome of infections or autoimmunity. Type I (also called invariant) NKT cells (iNKT) are best characterized mainly because of the availability of lipid antigen-loaded CD1d-tetramer detection reagents. Human iNKT cells present important phenotypic differences relative to their murine counterpart, restricting the extrapolation of findings from experimental murine models to human health and disease states. Particularly, the ontogeny and early life phenotype of iNKT cells largely differ between human and mice, indicating divergent functional properties between species. The high therapeutic potential offered by manipulation of iNKT cells in disease warrants a better understanding of human iNKT cell biology. Here, we discuss characteristics of human iNKT cells and present an efficient and rapid method for their ex vivo purification and characterization.
| S-EPMC4619040 | biostudies-literature
Project description:The ex vivo generation of gastrointestinal organoids from crypt stem cells opens up the possibility of new research approaches investigating gastrointestinal diseases. We used this technology to study differences between healthy controls and patients with celiac disease (CD). We noticed distinct dissimilarities in the phenotypes of organoids between our study groups and found considerable variations in their gene expression. Extracellular matrix genes involved in epithelial-mesenchymal transition are expressed most differently. In addition, we demonstrated epigenetic modifications that might be responsible for the different organoid gene expression thus accounting for a deranged crypt/villus axis development in CD. The organoids have proven valuable to demonstrate fundamental differences in duodenal derived organoids between healthy controls and patients with CD and thus are a suitable tool to gain new insights in pathogenesis of CD.
| S-EPMC7044285 | biostudies-literature
Project description:Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.
| S-EPMC4769231 | biostudies-literature
Project description:In this study, we aimed to detect and characterize ex vivo virus-specific CD8+ T cells in patients with immune-tolerant hepatitis B virus (HBV) infection. We investigated a Korean chronic hepatitis B cohort composed of 15 patients in the immune-tolerant phase, 17 in the immune-active phase, and 13 under antiviral treatment. We performed enzyme-linked immunospot (ELISpot) assays ex vivo and intracellular cytokine staining after in vitro culture. We also performed ex vivo multimer staining assays and examined the expression of programmed death-1 (PD-1) and CD127 in pentamer-positive cells. Ex vivo ELISpot revealed that HBV-specific T cell function was weaker in immune-tolerant patients than in those under antiviral treatment. In vitro culture of peripheral blood mononuclear cells for 10 days revealed that HBV-specific CD8+ T cells produced interferon-γ in some immune-tolerant patients. We detected HBV-specific CD8+ T cells ex vivo (using the HBV core18-27 pentamer) in patients from all three groups. The PD-1+ subset of pentamer+ CD8+ T cells was smaller ex vivo in the immune-tolerant phase than in the immune-active phase or under antiviral treatment. Interestingly, the proportion of PD-1+ CD8+ T cells in HBV-specific CD8+ T cells correlated with patient age when all enrolled patients were analyzed. Overall, HBV-specific CD8+ T cells are present in patients considered as immune-tolerant, although their ex vivo functionality is significantly weaker than that in patients under antiviral treatment (P < 0.05). Despite the high viral load, the proportion of PD-1 expression in HBV-specific CD8+ T cells is lower in the immune-tolerant phase than in other phases. Our results indicate appropriate stimulation may enhance the effector function of HBV-specific CD8+ T cells in patients considered as being in the immune-tolerant phase.
| S-EPMC6563765 | biostudies-literature
Project description:BackgroundRegulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB.MethodWe isolated PBMC from smear positive TB patients (TB, N = 13) before and after treatment, latent TB infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers.ResultsThe findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4(+)CD25(+/hi) T cells was higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4(+)CD25(+)FOXP3(+)CD127(lo) Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4(+)CD25(+)FOXP3(+)CD127(lo)CD45RO(+)Ki-67(+)) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO(+)Ki-67(-)) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5-21.9) of Tregs to 9.3% (7.0-12.2); P = 0.003 Wilcoxon signed rank test. We conclude that Treg subsets may be differentially regulated and expressed in TB disease, cure, and infection.
| S-EPMC5111553 | biostudies-literature
Project description:The increase in life expectancy and the migration of individuals with Chagas disease (ChD) from rural to urban centers exposes them to the development of chronic-degenerative abnormalities that may increase the prevalence of metabolic syndrome (MetS). The present study aimed to identify the prevalence of MetS and its components in individuals with chronic ChD. This is a cross-sectional study with 361 patients of both sexes, aging >18 years, followed at a national reference center (Rio de Janeiro, Brazil). MetS diagnosis followed the International Diabetes Federation 2005 criteria. The association between the variables was determined through logistic regression models. The mean age was and 60.7±10.8 years. About half (56.2%) were female and the majority self-reported their race as mulatto (59.8%). The percentage of individuals with MetS was 40.4%. The variables independently associated with MetS were age (OR 1.06; 95%CI 1.04-1.09), high education levels (OR 0.36; 95%CI 0.17-0.79) and cardiac form with heart failure (OR 0.34; 95%CI 0.17-0.68). Therefore, a high prevalence of MetS was found in this Brazilian chronic ChD cohort. The identification of the associated factors can facilitate the development of effective approaches for preventing and managing MetS in ChD patients.
| S-EPMC8018626 | biostudies-literature