Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous ...assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.
The metabolic changes that occur in tumor microenvironment (TME) can influence not only the biological activity of tumor cells, which become more aggressive and auto sustained, but also the immune ...response against tumor cells, either producing ineffective responses or polarizing the response toward protumor activity. γδ T cells are a subset of T cells characterized by a plasticity that confers them the ability to differentiate towards different cell subsets according to the microenvironment conditions. On this basis, we here review the more recent studies focused on altered tumor metabolism and γδ T cells, considering their already known antitumor role and the possibility of manipulating their effector functions by in vitro and in vivo approaches. γδ T cells, thanks to their unique features, are themselves a valid alternative to overcome the limits associated with the use of conventional T cells, such as major histocompatibility complex (MHC) restriction, costimulatory signal and specific tumor-associated antigen recognition. Lipids, amino acids, hypoxia, prostaglandins and other metabolic changes inside the tumor microenvironment could reduce the efficacy of this important immune population and polarize γδ T cells toward IL17 producing cells that play a pro tumoral role. A deeper knowledge of this phenomenon could be helpful to formulate new immunotherapeutic approaches that target tumor metabolisms.
T-cell-mediated immune responses play a fundamental role in controlling
(
) infection, and traditionally, this response is thought to be mediated by Th1-type CD4
T-cells secreting IFN-γ. While ...studying the function and specificity of
-reactive CD4
T-cells in more detail at the single cell level; however, we found a human CD4
T-cell population with a naive phenotype that interestingly was capable of producing multiple cytokines (T
cells). CD4
T
cells phenotyped as CD95
CD28
CD49d
CXCR3
and showed a broad distribution of T cell receptor Vβ segments. They rapidly secreted multiple cytokines in response to different
antigens, their frequency was increased during active disease, but was comparable to latent tuberculosis infection in treated TB patients. These results identify a novel human CD4
T-cell subset involved in the human immune response to mycobacteria, which is present in active TB patients' blood. These results significantly expand our understanding of the immune response in infectious diseases.
Inflammatory bowel disease (IBD) is an increasingly urgent medical problem that strongly impairs quality of life for patients. A global rise in incidence has been observed over the last few decades, ...with the highest incidence rates recorded in North America and Europe. Still, an increased incidence has been reported in the last ten years in newly industrialized countries in Asia, including China and India, both with more than one billion inhabitants. These data underline that IBD is an urgent global health problem. In addition, it is estimated that between 20% and 30% of IBD patients will develop colorectal cancer (CRC) within their lifetime and CRC mortality is approximately 50% amongst IBD patients. Although the exact etiology of IBD is still being defined, it is thought to be due to a complex interaction between many factors, including defects in the innate and adaptive immune system; microbial dysbiosis, i.e., abnormal levels of, or abnormal response to, the gastrointestinal microbiome; a genetic predisposition; and several environmental factors. At present, however, it is not fully understood which of these factors are the initiators of inflammation and which are compounders. The purpose of this review is to analyze the complex balance that exists between these elements to maintain intestinal homeostasis and prevent IBD or limit adverse effects on people's health.
Glial tumors alone account for 40% of all CNS tumors and present a low survival rate. The tumor microenvironment is a critical regulator of tumor progression and therapeutic effectiveness in glioma. ...Growing evidence from numerous studies of human solid tumor-infiltrating CD8+ T cells indicates that tissue-resident memory T cells (TRM) represent a substantial subpopulation of tumor-infiltrating lymphocytes (TILs). Although it is reported that some types of cancer patients with high immune infiltration tend to have better outcomes than patients with low immune infiltration, it seems this does not happen in gliomas. This study aimed to characterize TRMs cells in the glioma tumor microenvironment to identify their potential predictive and prognostic role and the possible therapeutic applications. Fluorescence activated cell sorting (FACS) analysis and immunofluorescence staining highlighted a statistically significant increase in CD8+ TRM cells (CD103+ and CD69+ CD8+ T cells) in gliomas compared to control samples (meningioma). In-silico analysis of a dataset of n = 153 stage IV glioma patients confirmed our data. Moreover, the gene expression analysis showed an increase in the expression of TRM-related genes in tumor tissues compared to normal tissues. This analysis also highlighted the positive correlation between genes associated with CD8+ TRM and TILs, indicating that CD8+ TRMs cells are present among the infiltrating T cells. Finally, high expression of Integrin subunit alpha E (ITGAE), the gene coding for the integrin CD103, and high CD8+ TILs abundance were associated with more prolonged survival, whereas high ITGAE expression but low CD8+ TILs abundance were associated with lower survival.
We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a ...strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca2+ influx, (iii) promote Ca2+-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca2+-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1β, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-β without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1β, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.
Tuberculosis (TB) remains one of the most important health challenges worldwide. Control of the TB epidemic has not yet been achieved because of the lack of an effective vaccine and rapid and ...sensitive diagnostic approaches, as well as the emergence of drug-resistant forms of
. Cellular immunity has a pivotal role against
infection, but the role of humoral immunity is still controversial. We analyzed the frequency, absolute counts, and phenotypic and functional subsets of B lymphocytes in the peripheral blood of patients with active TB and subjects with latent infection compared to healthy donors. Moreover, we analyzed serum levels of total Ig and their IgA, IgM, and IgG isotypes and the titers of preexisting antibodies against a pool of common viral pathogens. FlowCT and unsupervised clusterization analysis show that patients with active TB and LTBI subjects have modest non-significant reduction in the numbers of circulating B lymphocytes as compared to healthy donors. Moreover, LTBI subjects had high percentages of atypical B cell population and lower percentages of naive and switched memory B cells. These findings were supported by gene expression and GSEA analysis. Moreover, there were no differences between active TB patients, LTBI subjects and HD, either in serum levels of total Ig isotypes or in preexisting IgG antibody titers, to ten different antigens from eight common pathogenic viruses, clearly demonstrating that either active or latent
infection preserves the antibody production capacity of long-lived plasma cells. Thus, our results agree with previous studies reporting unaltered B cell frequencies in the blood of active TB patients and LTBI individuals as compared to healthy controls.
(1) Background: statins have been considered an attractive class of drugs in the pharmacological setting of COVID-19 due to their pleiotropic properties and their use correlates with decreased ...mortality in hospitalized COVID-19 patients. Furthermore, it is well known that statins, which block the mevalonate pathway, affect γδ T lymphocyte activation. As γδ T cells participate in the inflammatory process of COVID-19, we have investigated the therapeutical potential of statins as a tool to inhibit γδ T cell pro-inflammatory activities; (2) Methods: we harvested peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild clinical manifestations, COVID-19 recovered patients, and healthy controls. We performed ex vivo flow cytometry analysis to study γδ T cell frequency, phenotype, and exhaustion status. PBMCs were treated with Atorvastatin followed by non-specific and specific stimulation, to evaluate the expression of pro-inflammatory cytokines; (3) Results: COVID-19 patients had a lower frequency of circulating Vδ2+ T lymphocytes but showed a pronounced pro-inflammatory profile, which was inhibited by in vitro treatment with statins; (4) Conclusions: the in vitro capacity of statins to inhibit Vδ2+ T lymphocytes in COVID-19 patients highlights a new potential biological function of these drugs and supports their therapeutical use in these patients.
In this paper, we present demographic, clinical, anamnestic, cognitive, and functional data, as well as haematological, haematochemical, immunological, and genetic parameters of an exceptional ...individual: A.T., a semi-supercentenarian who held the title of the oldest living Italian male centenarian from 28 December 2020, to 23 September 2021. The purpose of this study is to provide fresh insights into extreme phenotypes, with a particular focus on immune-inflammatory parameters. To the best of our knowledge, this study represents the first phenotypic investigation of a semi-supercentenarian, illustrating both INFLA-score, a metric designed to assess the cumulative impact of inflammatory markers and indicators of age-related immune phenotype (ARIP), recognized as significant gauges of biological ageing. The aim of this study was, indeed, to advance our understanding of the role of immune-inflammatory responses in achieving extreme longevity. The results of laboratory tests, as well as clinical history and interview data, when compared to the results of our recent study on Sicilian centenarians, demonstrate an excellent state of health considering his age. Consistent with previous studies, we observed increased IL-6 inflammatory markers and INFLA score in A.T. More interestingly, the semi-supercentenarian showed values of ARIP indicators such as naïve CD4+ cells, CD4+/CD8+ ratio, and CD4+TN/TM ratio in the range of young adult individuals, suggesting that his immune system's biological age was younger than the chronological one. The results support the notion that the immune system can play a role in promoting extreme longevity. However, this does not rule out the involvement of other body systems or organs in achieving extreme longevity.
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