232 research outputs found
Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from mycobacterium tuberculosis
A number of different interferon-c ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are
transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity.
Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-c spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen
Atypical Human Effector/Memory CD4+ T Cells With a Naive-Like Phenotype
The induction of adaptive immunological memory, mediated by T and B cells, plays an important role in protective immunity to pathogens induced by previous infections or vaccination. Naive CD4+ T cells that have been primed by antigen develop into memory or effector cells, which may be distinguished by their capability to exert a long-term and rapid response upon re-challenge by antigen, to produce distinct cytokines and surface marker expression phenotypes such as CD45RA/RO, CD27, CD62L, and CCR7. Moreover, a distinct lineage of memory T cells populates tissues (tissue-resident memory T cells or TRM cells) which orchestratea the response to pathogens re encountered at tissue sites. Recent evidence, however, has highlighted that CD4+ naive T cells are much more heterogeneous that previously thought, and that they harbor diversity in phenotypes, differentiation stages, persistence, functions, and anatomic localizations. These cells represent cellular subsets that are extremely heterogeneous and multifunctional at their very initial stages of differentiation, with the potential to become âatypicalâ memory and effector cells. In this mini review, we focus on recently obtained data from studies in humans, in which this newly recognized heterogeneity in the naive T cell pool was discovered in terms of surface marker expression, cytokine production, or transcriptomic profiles. The deep analysis of immune functions at the single cell level combined with a better understanding of the generation and maintenance of the various atypical memory CD4+ T cell subsets with a naive-like phenotype will be important in immune-monitoring of vaccination and immunotherapies in infectious diseases
Human CD4 T-cells with a naive phenotype produce multiple cytokines during Mycobacterium tuberculosis infection and correlate with active disease
T-cell-mediated immune responses play a fundamental role in controlling Mycobacterium tuberculosis (M. tuberculosis) infection, and traditionally, this response is thought to be mediated by Th1-type CD4+ T-cells secreting IFN-Îł. While studying the function and specificity of M. tuberculosis-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 (TCNP cells). CD4+ TCNP cells phenotyped as CD95lo CD28int CD49dhi CXCR3hi and showed a broad distribution of T cell receptor VÎČ segments. They rapidly secreted multiple cytokines in response to different M. tuberculosis 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
Elderly Subjects Have a Delayed Antibody Response and Prolonged Viraemia following Yellow Fever Vaccination: A Prospective Controlled Cohort Study
Yellow fever vaccination (YF-17D) can cause serious adverse events (SAEs). The mechanism of these SAEs is poorly understood. Older age has been identified as a risk factor. We tested the hypothesis that the humoral immune response to yellow fever vaccine develops more slowly in elderly than in younger subjects.We vaccinated young volunteers (18â28 yrs, Nâ=â30) and elderly travelers (60â81 yrs, Nâ=â28) with YF-17D and measured their neutralizing antibody titers and plasma YF-17D RNA copy numbers before vaccination and 3, 5, 10, 14 and 28 days after vaccination.â=â0.02, using a mixed linear model. Viraemia was more common in the elderly (86%, 24/28) than in the younger participants (60%, 14/30) (pâ=â0.03) with higher YF-17D RNA copy numbers in the elderly participants.We found that elderly subjects had a delayed antibody response and higher viraemia levels after yellow fever primovaccination. We postulate that with older age, a weaker immune response to yellow fever vaccine allows the attenuated virus to cause higher viraemia levels which may increase the risk of developing SAEs. This may be one piece in the puzzle of the pathophysiology of YEL-AVD
Thyrotrophin and thyroxine support immune homeostasis in humans
The endocrine and the immune systems interact by sharing receptors for hormones and cytokines, cross-control and feedback mechanisms. To date, no comprehensive study has assessed the impact of thyroid hormones on immune homeostasis. By studying immune phenotype (cell populations, antibody concentrations, circulating cytokines, adipokines and acute-phase proteins, monocyte-platelet interactions and cytokine production capacity) in two large independent cohorts of healthy volunteers of Western European descent from the Human Functional Genomics Project (500FG and 300BCG cohorts), we identified a crucial role of the thyroid hormone thyroxin (T4) and thyroid-stimulating hormone (TSH) on the homeostasis of lymphocyte populations. TSH concentrations were strongly associated with multiple populations of both effector and regulatory T cells, whereas B-cell populations were significantly associated with free T4 (fT4). In contrast, fT4 and TSH had little impact on myeloid cell populations and cytokine production capacity. Mendelian randomization further supported the role of fT4 for lymphocyte homeostasis. Subsequently, using a genomics approach, we identified genetic variants that influence both fT4 and TSH concentrations and immune responses, and gene set enrichment pathway analysis showed enrichment of fT4-affected gene expression in B-cell function pathways, including the CD40 pathway, further supporting the importance of fT4 in the regulation of B-cell function. In conclusion, we show that thyroid function controls the homeostasis of the lymphoid cell compartment. These findings improve our understanding of the immune responses and open the door for exploring and understanding the role of thyroid hormones in the lymphocyte function during disease
Pro-inflammatory Monocyte Phenotype During Acute Progression of Cerebral Small Vessel Disease
Background: The etiology of cerebral small vessel disease (SVD) remains elusive, though evidence is accumulating that inflammation contributes to its pathophysiology. We recently showed retrospectively that pro-inflammatory monocytes are associated with the long-term progression of white matter hyperintensities (WMHs). In this prospective high-frequency imaging study, we hypothesize that the incidence of SVD progression coincides with a pro-inflammatory monocyte phenotype.
Methods: Individuals with SVD underwent monthly magnetic resonance imaging (MRI) for 10 consecutive months to detect SVD progression, defined as acute diffusion-weighted imaging-positive (DWI+) lesions, incident microbleeds, incident lacunes, and WMH progression. Circulating inflammatory markers were measured, cytokine production capacity of monocytes was assessed after ex vivo stimulation, and RNA sequencing was performed on isolated monocytes in a subset of participants.
Results: 13 out of 35 individuals developed SVD progression (70 ± 6 years, 54% men) based on incident lesions (n = 7) and/or upper quartile WMH progression (n = 9). Circulating E-selectin concentration (p < 0.05) and the cytokine production capacity of interleukin (IL)-1ÎČ and IL-6 (p < 0.01) were higher in individuals with SVD progression. Moreover, RNA sequencing revealed a pro-inflammatory monocyte signature including genes involved in myelination, bloodâbrain barrier, and endothelialâleukocyte interaction.
Conclusions: Circulating monocytes of individuals with progressive SVD have an inflammatory phenotype, characterized by an increased cytokine production capacity and a pro-inflammatory transcriptional signature
The Genetic Risk for COVID-19 Severity Is Associated With Defective Immune Responses
Recent genome-wide association studies (GWASs) of COVID-19 patients of European ancestry have identified genetic loci significantly associated with disease severity. Here, we employed the detailed clinical, immunological and multi-omics dataset of the Human Functional Genomics Project (HFGP) to explore the physiological significance of the host genetic variants that influence susceptibility to severe COVID-19. A genomics investigation intersected with functional characterization of individuals with high genetic risk for severe COVID-19 susceptibility identified several major patterns: i. a large impact of genetically determined innate immune responses in COVID-19, with ii. increased susceptibility for severe disease in individuals with defective cytokine production; iii. genetic susceptibility related to ABO blood groups is probably mediated through the von Willebrand factor (VWF) and endothelial dysfunction. We further validated these identified associations at transcript and protein levels by using independent disease cohorts. These insights allow a physiological understanding of genetic susceptibility to severe COVID-19, and indicate pathways that could be targeted for prevention and therapy
Serum Biomarker Profile Including CCL1, CXCL10, VEGF, and Adenosine Deaminase Activity Distinguishes Active From Remotely Acquired Latent Tuberculosis
INTRODUCTION: There is an urgent medical need to differentiate active tuberculosis (ATB) from latent tuberculosis infection (LTBI) and prevent undertreatment and overtreatment. The aim of this study was to identify biomarker profiles that may support the differentiation between ATB and LTBI and to validate these signatures. MATERIALS AND METHODS: The discovery cohort included adult individuals classified in four groups: ATB (n = 20), LTBI without prophylaxis (untreated LTBI; n = 20), LTBI after completion of prophylaxis (treated LTBI; n = 20), and healthy controls (HC; n = 20). Their sera were analyzed for 40 cytokines/chemokines and activity of adenosine deaminase (ADA) isozymes. A prediction model was designed to differentiate ATB from untreated LTBI using sparse partial least squares (sPLS) and logistic regression analyses. Serum samples of two independent cohorts (national and international) were used for validation. RESULTS: sPLS regression analyses identified C-C motif chemokine ligand 1 (CCL1), C-reactive protein (CRP), C-X-C motif chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) as the most discriminating biomarkers. These markers and ADA(2) activity were significantly increased in ATB compared to untreated LTBI (p †0.007). Combining CCL1, CXCL10, VEGF, and ADA2 activity yielded a sensitivity and specificity of 95% and 90%, respectively, in differentiating ATB from untreated LTBI. These findings were confirmed in the validation cohort including remotely acquired untreated LTBI participants. CONCLUSION: The biomarker signature of CCL1, CXCL10, VEGF, and ADA2 activity provides a promising tool for differentiating patients with ATB from non-treated LTBI individuals
Correction to: Rewiring of glucose metabolism defines trained immunity induced by oxidized low-density lipoprotein
The correct name of the 17th Author is presented in this paper. In the paragraph âMetabolic analysisâ of the Method section âan XFp Analyzerâ should be changed to âan XFe96 Analyzerâ.</p
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