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Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

ABSTRACT: Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFN?, TNF?, IL2 and MIP1? production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.

SUBMITTER: Kutscher S

PROVIDER: S-EPMC3795753 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

Kutscher Sarah S Dembek Claudia J CJ Deckert Simone S Russo Carolina C Körber Nina N Bogner Johannes R JR Geisler Fabian F Umgelter Andreas A Neuenhahn Michael M Albrecht Julia J Cosma Antonio A Protzer Ulrike U Bauer Tanja T

PloS one 20131011 10

Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV- ...[more]

PMID: 24146841

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Project description:BackgroundIn the poorly understood relationship between orthostatic hypotension and falls, next to blood pressure (BP), baroreflex sensitivity (BRS) and cerebral autoregulation (CAR) may be key measures. The posture- and movement dependency of orthostatic hypotension requires continuous and unobtrusive monitoring. This may be possible using simultaneous photoplethysmography (PPG), electrocardiography (ECG), and near-infrared spectroscopy (NIRS) signal recordings, from which pulse wave velocity (PWV; potentially useful for BP estimation), BRS and CAR can be derived. The PPG, NIRS and PWV signal correlation with BP and BRS/CAR reliability and validity need to be addressed.MethodsIn 34 healthy adults (mean age 25 years, inter quartile range 22-45; 10 female), wrist and finger PPG, ECG, bifrontal NIRS (oxygenated and deoxygenated hemoglobin) and continuous BP were recorded during sit to stand and supine to stand movements. Sixteen participants performed slow and rapid supine to stand movements; eighteen other participants performed a 1-min squat movement. Pulse wave velocity (PWV) was defined as the inverse of the ECG R-peak to PPG pulse delay; PPG, NIRS and PWV signal correlation with BP as their Pearson correlations with mean arterial pressure (MAP) within 30 s after the postural changes; BRS as inter beat interval drop divided by systolic BP (SBP) drop during the postural changes; CAR as oxygenated hemoglobin drop divided by MAP drop. BRS and CAR were separately computed using measured and estimated (linear regression) BP. BRS/CAR reliability was defined by the intra class correlation between repeats of the same postural change; validity as the Pearson correlation between BRS/CAR values based on measured and estimated BP.ResultsThe highest correlation with MAP was found for finger PPG and oxygenated hemoglobin, ranging from 0.75-0.79 (sit to stand), 0.66-0.88 (supine to stand), and 0.82-0.94 (1-min squat). BRS and CAR reliability was highest during the different supine to stand movements, ranging from 0.17 - 0.49 (BRS) and 0.42-0.75 (CAR); validity was highest during rapid supine to stand movements, 0.54 and 0.79 respectively.ConclusionPPG-ECG-NIRS recordings showed high correlation with BP and enabled computation of reliable and valid BRS and CAR estimates, suggesting their potential for continuous unobtrusive monitoring of orthostatic hypotension key measures.

Project description:While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.

Project description:BackgroundMALDI-TOF mass spectrometry (MS) on whole cells enables the detection of different cell types and cell activation. Here, we wondered whether this approach would be useful to investigate the host response in sepsis.MethodsPeripheral blood mononuclear cells (PBMCs) from patients with severe sepsis and healthy donors were analyzed with MALDI-TOF MS. PBMCs from healthy donors were also stimulated with lipopolysaccharide, peptidoglycan, CpG oligonucleotides, polyinosinic polycytidylic acid, and with heat-inactivated bacteria. Averaged spectra of PBMCs stimulated in vitro by different agonists were generated from the database using the Biotyper software and matching scores between each spectrum from patients and averaged spectra from the database were calculated.ResultsWe show that the MALDI-TOF MS signature of PBMCs from septic patients was specific, compared with healthy controls. As the fingerprints observed in patients may be related to PBMC activation, PBMCs from healthy controls were stimulated with cytokines, soluble Pathogen-Associated Molecular Patterns (PAMPs) and heat-killed bacteria, and we created a database of reference spectra. The MALDI-TOF MS profiles of PBMCs from septic patients were then compared with the database. No differences were found between patients with documented infection (n = 6) and those without bacteriological documentation (n = 6). The spectra of PBMCs from septic patients matched with those of interferon-γ- and interleukin-10-stimulated PBMCs, confirming that sepsis is characterized by both inflammatory and immunoregulatory features. Interestingly, the spectra of PBMCs from septic patients without documented infection matched with the reference spectrum of PBMCs stimulated by CpG oligonucleotides, suggesting a bacterial etiology in these patients.ConclusionsDespite the limits of this preliminary study, these results indicate that the monitoring of functional status of PBMCs in peripheral blood by whole cell MALDI-TOF MS could provide unique opportunities to assess disease progression or resolution in clinical settings.

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Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

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Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.

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