Whole Blood Flow Cytometry Assay Research Articles

Altered Ex Vivo NLRP3 Inflammasome Activation Is Associated with 28-Day Mortality in Septic Patients.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated response to infection. In this context, the aberrant activation of the NLRP3 inflammasome has been documented mostly through the measurement of increased plasmatic concentrations of IL-1β and IL-18. At the cellular level, contradictory results have been published. However, no study has comprehensively monitored NLRP3 inflammasome activation at the basal level and after ex vivo reactivation of whole blood monocytes and neutrophils focusing on ICU patients with bacterial and viral sepsis, including a longitudinal analysis. Thus, we conducted a prospective longitudinal study, examining NLRP3 inflammasome functionality in COVID-19 ICU patients (n = 15) and bacterial septic shock patients (n = 17) during the first week of ICU hospitalization, compared with healthy donors. Using two whole-blood flow cytometry assays, we detected ASC speck-positive monocytes (i.e., monocytes presenting the polymerization of ASC proteins) and activated caspase-1 in polymorphonuclear cells as read-outs, both at baseline and following nigericin stimulation, a drug that forms pores and activates the NLRP3 inflammasome. Our findings showed that, at baseline and regardless of the type of infection, patients exhibited reduced ASC speck-positive monocytes and decreased activated caspase-1 in PMN compared to healthy volunteers. This decrease was prominent at day 0. Following nigericin stimulation, this reduction was also observed and persisted throughout the first week of hospitalization, irrespective of the cellular population or parameter being considered. Notably, at day 0, this diminished activation and response to stimulation of NLRP3 was associated with a higher 28-day mortality rate. Consequently, our observations highlighted a concurrent decline in both basal expression and ex vivo activation of the NLRP3 inflammasome in circulating myeloid cells from patients with bacterial and viral sepsis in association with increased mortality.

SARS-CoV-2-reactive IFN-γ-producing CD4+ and CD8+ T cells in blood do not correlate with clinical severity in unvaccinated critically ill COVID-19 patients

We examined the relationship between peripheral blood levels of SARS-CoV-2 S (Spike protein)1/M (Membrane protein)-reactive IFN-γ-producing CD4+ and CD8+ T cells, serum levels of biomarkers of clinical severity, and mortality in critically ill COVID-19 patients. The potential association between SARS-CoV-2-S-Receptor Binding Domain (RBD)-specific IgG levels in sera and mortality was also investigated. SARS-CoV-2 T cells and anti-RBD IgG levels were monitored in 71 non-consecutive patients (49 male and 22 female; median age, 65 years) by whole-blood flow cytometry and Enzyme-linked immunosorbent assay (ELISA), respectively (326 specimens). SARS-CoV-2 RNA loads in paired tracheal aspirates [TA] (n = 147) were available from 54 patients. Serum levels of interleukin-6, ferritin, D-Dimer, lactose dehydrogenase and C-reactive protein in paired sera were known. SARS-CoV-2 T cells (either CD4+, CD8+ or both) were detectable in 70 patients. SARS-CoV-2 IFN-γ CD4+ T-cell responses were documented more frequently than their CD8+ counterparts (62 vs. 56 patients) and were of greater magnitude overall. Detectable SARS-CoV-2 S1/M-reactive CD8+ and CD4+ T-cell responses were associated with higher SARS-CoV-2 RNA loads in TA. SARS-CoV-2 RNA load in TA decreased over time, irrespective of the dynamics of SARS-CoV-2-reactive CD8+ and CD4+ T cells. No correlation was found between SARS-CoV-2 IFN-γ T-cell counts, anti-RBD IgG concentrations and biomarker serum levels (Rho ≤ 0.3). The kinetics of both T cell subsets was comparable between those who died or survived, whereas anti-RBD IgG levels were higher across different time points in deceased patients than in survivors. Enumeration of peripheral blood levels of SARS-CoV-2-S1/M-reactive IFN-γ CD4+ and CD8+ T cells does not predict viral clearance from the lower respiratory tract or poor clinical outcomes in critically ill COVID-19 patients. In contrast, anti-RBD IgG levels were directly associated with increased mortality.

Human Immunodeficiency Virus Infection Impairs Th1 and Th17 Mycobacterium tuberculosis–Specific T-Cell Responses

Human immunodeficiency virus (HIV)-infected individuals have a higher risk of developing active tuberculosis (TB) than HIV-uninfected individuals, but the mechanisms underpinning this are unclear. We hypothesized that depletion of specific components of Mycobacterium tuberculosis (Mtb)-specific CD4+ and CD8+ T-cell responses contributed to this increased risk. Mtb-specific T-cell responses in 147 HIV-infected and 44 HIV-uninfected control subjects in a TB-endemic setting in Bloemfontein, South Africa, were evaluated. Using a whole-blood flow cytometry assay, we measured expression of interferon gamma, tumor necrosis factor alpha, interleukin 2, and interleukin 17 in CD4+ and CD8+ T cells in response to Mtb antigens (PPD, ESAT-6/CFP-10 [EC], and DosR regulon-encoded α-crystallin [Rv2031c]). Fewer HIV-infected individuals had detectable CD4+ and CD8+ T-cell responses to PPD and Rv2031c than HIV-uninfected subjects. Mtb-specific T cells showed distinct patterns of cytokine expression comprising both Th1 (CD4 and CD8) and Th17 (CD4) cytokines, the latter at highest frequency for Rv2031c. Th17 antigen-specific responses to all antigens tested were specifically impaired in HIV-infected individuals. HIV-associated impairment of CD4+ and CD8+Mtb-specific T-cell responses is antigen specific, particularly impacting responses to PPD and Rv2031c. Preferential depletion of Th17 cytokine-expressing CD4+ T cells suggests this T-cell subset may be key to TB susceptibility in HIV-infected individuals.

Comparison of FcRγ-Deficient and CD57+ Natural Killer Cells Between Cord Blood and Adult Blood in the Cytomegalovirus-Endemic Korean Population.

BackgroundFcRγ-deficient natural killer (NK) cells (g-NK cells) have been associated with cytomegalovirus (CMV) infection. However, the frequency of g-NK cells in a CMV-endemic area (i.e., Korea) has not yet been studied. We examined the frequency of g-NK cells and expression of CD57 on NK cells in cord blood (CB) and adult blood (AB).MethodsOf the 24 AB samples collected, 95.8% (23/24) were CMV IgG+/IgM-, while 100% of the 13 healthy CB samples were CMV IgG+/IgM-. We performed whole-blood flow cytometry assays to analyze intracellular FcRγ and CD3ζ expression of CD3-/CD56dim NK cells from 13 CB and 24 AB samples, and surface CD57 expression on CD3-/CD56dim/CD16+ NK cells from 13 CB and 19 AB samples.ResultsAll CMV seropositive AB samples contained g-NK cells (23/23), and the median proportion of g-NK cells in the CD3-/CD56dim NK cell pool was 35.0% (range: 11-77%). CD57+ NK cells in the CD3-/CD56dim/CD16+ NK cell population were detected in all 19 AB samples tested, but not in any CB samples.ConclusionsOur data suggest that g-NK cells and CD57+ NK cells are present at a very high frequency in CMV-seropositive AB, but rare in CMV-naïve CB.

Investigating the role of rare genetic variants of GP6 gene in platelet function

BackgroundThrombus formation in the arteries is a major risk factor for myocardial infarction and stroke. Platelets are involved in blood homoeostasis because of their ability to form thrombi after the interaction with collagen. Glycoprotein VI (GPVI) in complex with the Fc receptor γ chain forms the major collagen receptor on the platelet membrane. Clustering of the receptor on collagen or collagen-related peptides (CRP-XL) triggers the kinase activity of GPVI which leads to various platelet responses that can be measured in vitro. We aimed to identify and characterise differences in GPVI expression and function in individuals with rare GPVI phenotypes with a view to uncover potentially causal genetic variants. In addition, we tested the hypothesis that individuals with both extremely low GPVI expression and responses to CRP-XL could have previously undescribed genomic sequence variation in the GP6 gene, which encodes the GPVI protein receptor. MethodsWe measured surface glycoprotein expression in 506 healthy individuals with flow cytometry using an anti-GPVI monoclonal antibody. Total glycoprotein expression was measured with western blotting using rabbit anti-GPVI polyclonal antibodies. Flow cytometry was used to measure platelet surface expression of GPVI. Thrombus formation was assessed by perfusing heparinised blood over a collagen-coated glass slide followed by calculation of the area covered by thrombi. FindingsTwo individual showed significantly reduced expression of GPVI, impaired responses to CRP-XL, but normal responses to ADP. The low platelet expression level of GPVI in these two individuals was not balanced by an increase in plasma GPVI, ruling out a metalloproteinase-dependent shedding effect. Sequencing the promoter and the exons of GP6 in these two individuals showed a non-synonymous single nucleotide polymorphism (SNP) in exon 4 of the GP6 gene in each person—in one, a S195N substitution, which is a rare deleterious mutation, and in the other, a previously undocumented SNP in the same region leading to a P194T substitution. Studies in this latter individual, together with three siblings and a son, showed a range of expression levels consistent with their GP6 haplotype status, with no expression from the variant-containing allele. Carriers of the rare variant showed a relatively mild reduction in thrombus formation compared with controls. InterpretationOur screening of GPVI-related phenotypes has identified deficiencies in this important platelet receptor in a pedigree of healthy individuals. Their lack of obvious bleeding problems can partly be explained by the observation that alternative activation pathways (thrombin receptors, ADP receptors) function well in the whole-blood flow cytometry assay and would compensate in normal haemostasis. Further investigations to uncover the mechanisms giving rise to the lack of GPVI expression from this allele are continuing. FundingWellcome Trust, NHS Blood and Transplant.

Comparative analysis of whole-blood interferon-γ and flow cytometry assays for detecting post-treatment Immune responses in patients with active tuberculosis

Intracellular cytokine flow cytometry (ICCFC) has been explored to detect tuberculosis (TB) infections; however, there are little data regarding its use to examine the dynamic responses of Mycobacterium tuberculosis (MTB)-specific T-cells after anti-tuberculous therapy. The aim of this study was to analyze both dynamic changes in functional MTB antigen-specific T-cell subsets and interferon-gamma (IFN-γ) levels using ICCFC and the QuantiFERON-TB Gold In-Tube (QFT-IT) test, respectively, following anti-tuberculous treatment in patients with active TB.Twenty-six patients with active TB were enrolled in the study, and QFT-IT and ICCFC were performed simultaneously both before and after treatment. IFN-γ levels (QFT-IT test) and the numbers of IFN-γ- or tumor necrosis factor-alpha (TNF-α)-expressing T-cells (ICCFC assay) were examined after stimulation with MTB antigen.There was no significant reduction in the mean IFN-γ concentrations measured by the QFT-IT test after anti-tuberculous treatment (P = 0.314). ICCFC analysis showed that the numbers of IFN-γ(+) /CD4(-) T-cells, and CD4(+) T-cells producing TNF-α, either alone or in combination with IFN-γ, were significantly reduced after anti-tuberculous treatment. The IFN-γ(+) /TNF-α(+) /CD4(+) T-cell subset showed the greatest difference between untreated and treated patients with active TB (area under the curve = 0.734, P = 0.004).Unlike the QFT-IT test, ICCFC provides diverse immunological information about dynamic changes in the number of MTB antigen-specific T-cells following anti-tuberculous therapy. Thus, analysis of MTB antigen-stimulated T-cell responses using ICCFC might have a role to play in monitoring treatment responses in patients with active TB.

Comparative analysis of whole-blood interferon-γ and flow cytometry assays for detecting post-treatment immune responses in patients with active tuberculosis.

Background: Intracellular cytokine flow cytometry (ICCFC) has been explored to detect tuberculosis (TB) infections; however, there are little data regarding its use to examine the dynamic responses of Mycobacterium tuberculosis (MTB)-specific T-cells after anti-tuberculous therapy. The aim of the present study was to analyze both dynamic changes in functional MTB antigen-specific T-cell subsets and interferon-gamma (IFN-γ) levels using ICCFC and the QuantiFERON-TB Gold In-Tube (QFT-IT) test, respectively, following anti-tuberculous treatment in patients with active TB. Methods: Twenty-six patients with active TB were enrolled in the study, and QFT-IT and ICCFC were performed simultaneously both before and after treatment. IFN-γ levels (QFT-IT test) and the numbers of IFN-γ- or tumor necrosis factor-alpha (TNF-α)-expressing T-cells (ICCFC assay) were examined after stimulation with MTB antigen. Results: There was no significant reduction in the mean IFN-γ concentrations measured by the QFT-IT test after anti-tuberculous treatment (p=0.314). ICCFC analysis showed that the numbers of IFN-γ+ /CD4- T-cells and CD4+ T-cells producing TNF-α, either alone or in combination with IFN-γ, were significantly reduced after anti-tuberculous treatment. The IFN-γ+ /TNF-α+ /CD4+ T-cell subset showed the greatest difference between untreated and treated patients with active TB (area under the curve = 0.734, p=0.004). Conclusions: Unlike the QFT-IT test, ICCFC provides diverse immunological information about dynamic changes in the number of MTB antigen-specific T-cells following anti-tuberculous therapy. Thus, analysis of MTB antigen-stimulated T-cell responses using ICCFC might have a role to play in monitoring treatment responses in patients with active TB. © 2013 Clinical Cytometry Society.

HIV Infection Abrogates the Functional Advantage of Natural Killer Cells Educated through KIR3DL1/HLA-Bw4 Interactions To Mediate Anti-HIV Antibody-Dependent Cellular Cytotoxicity

Combinations of KIR3DL1 and HLA-Bw4 alleles protect against HIV infection and/or disease progression. These combinations enhance NK cell responsiveness through the ontological process of education. However, educated KIR3DL1(+) NK cells do not have enhanced degranulation upon direct recognition of autologous HIV-infected cells. Since antibody-dependent cellular cytotoxicity (ADCC) is associated with improved HIV infection outcomes and NK cells overcome inhibition through killer cell immunoglobulin-like receptors (KIR) to mediate ADCC, we hypothesized that KIR3DL1-educated NK cells mediate anti-HIV ADCC against autologous cells. A whole-blood flow cytometry assay was used to evaluate ADCC-induced activation of NK cells. This assay assessed activation (gamma interferon [IFN-γ] production and/or CD107a expression) of KIR3DL1(+) and KIR3DL1(-) NK cells, from HLA-Bw4(+) and HLA-Bw4(-) HIV-positive and HIV-negative individuals, in response to autologous HIV-specific ADCC targets. KIR3DL1(+) NK cells were more functional than KIR3DL1(-) NK cells from HLA-Bw4(+), but not HLA-Bw4(-), healthy controls. In HIV-infected individuals, no differences in NK cell functionality were observed between KIR3DL1(+) and KIR3DL1(-) NK cells in HLA-Bw4(+) individuals, consistent with dysfunction of NK cells in the setting of HIV infection. Reflecting the partial normalization of NK cell responsiveness following initiation of antiretroviral therapy, a significant correlation was observed between the peripheral CD4(+) T-lymphocyte counts in antiretroviral therapy-treated subjects and the functionality of NK cells. However, peripheral CD4(+) T-lymphocyte counts were not correlated with an anti-HIV ADCC functional advantage in educated KIR3DL1(+) NK cells. The abrogation of the functional advantage of educated NK cells may enhance HIV disease progression. Strategies to enhance the potency of NK cell-mediated ADCC may improve HIV therapies and vaccines.

Exercise training increases endothelial progenitor cells and decreases asymmetric dimethylarginine in peripheral arterial disease: A randomized controlled trial

BackgroundSupervised exercise training (SET) is recommended as initial treatment to improve walking capacity in peripheral arterial disease (PAD) patients with intermittent claudication. Various mechanisms by which SET yields beneficial effects are postulated, however data regarding its influence on angiogenesis are scarce. Thus, we designed a prospective randomized controlled trial to study the impact of SET on markers of angiogenesis and endothelial function in PAD. MethodsForty PAD patients were randomized to SET on top of best medical treatment (SET+BMT) for 6 months versus best medical treatment (BMT) only. Endothelial progenitor cells (EPC) were assessed by whole-blood flow cytometry (co-expression of CD34+ CD133+ KDR+) and cell culture assays (endothelial cell-colony forming units, circulating angiogenic cells, migration assay) at baseline, 3, 6 and 12-months after inclusion. Changes of plasma levels of asymmetric dimethylarginine (ADMA), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1) and maximum walking distance were determined. ResultsEPC – measured by flow cytometric and cell culture techniques – increased significantly upon training paralleled by a significant decrease of ADMA when compared to the BMT group (p<0.05). Six months after training cessation, the beneficial effect of SET on EPC diminished, but maximum walking distance was significantly improved compared to baseline and controls (p<0.05). No significant changes were observed for VEGF and SDF-1 plasma levels in time course. ConclusionsSET increases circulating EPC counts and decreases ADMA levels reflecting enhanced angiogenesis and improved endothelial function, which might contribute to cardiovascular risk reduction.

Abstract 5088: Exercise Training Increases Endothelial Progenitor Cells and Decreases Asymmetrical Dimethylarginine in Patients With Intermittent Claudication

Background - Exercise training is a well-established, non-invasive method to improve peripheral arterial perfusion and walking capacity in PAD patients. Hypothesis - We designed a prospective randomized controlled trial to test the effect of training on laboratory markers of angiogenesis. Methods - Thirty patients with intermittent claudication were randomized to supervised training twice a week for 6 months or standard medical therapy. Endothelial progenitor cells (EPC) were assessed by whole-blood flow cytometry (co-expression of CD34+CD133+KDR+) and cell culture assays (colony forming units/CFU and circulating angiogenic cells/CAC) at baseline, 3-months and 6-months after training as well as 12-months after study begin. Plasma ADMA was determined by immunoassay. In addition, changes in maximum walking distance were recorded. Results - EPC measured by flow cytometry increased significantly three and six months after training paralleled by a significant decrease of ADMA (asymmetric dimethylarginine), a major endogenous inhibitor of NO synthase (see table ). The number of CFUs and CACs were significantly higher after 6-months compared to baseline upon exercise (P&lt;0.05). No differences were observed in the standard group in timecourse. However, 6-months after training cessation the number of EPC and ADMA levels were similar to baseline. Exercise training showed a benefit on treadmill maximum walking distance with a trendwise increase at 6-months (training: baseline 116.6m±12.8 (mean±SEM) vs. 6-months 149.1m±17.8, P=0.08; standard: baseline 125.2m±23.9 vs. 6-months 108m±19.9, P=0.3). The benefit was sustained 6 months after training cessation in the exercise group (training: 12-months 157.5m±18.4, P=0.07; standard: 12-months 129.5m±26.9). Conclusions - The beneficial effect of exercise training in PAD patients might partly be due to EPC mobilization mediated by improved NO synthesis as we observed reduced ADMA levels. EPC and ADMA levels in timecourse

Hyper-IgM syndrome: report of one case.

The hyper-IgM syndrome (HIM) is a rare primary immunodeficiency disorder caused by defects in the CD40 ligand (CD40L)/CD40-signaling pathway. It is characterized by recurrent infections with markedly decreased IgG, IgA and IgE levels but normal or elevated serum IgM levels. A 5-month-old boy presented with rapidly progressive pneumonia which responded poorly to antibiotics. High levels of IgM and very low levels of IgG, IgE and IgA were noted in his plasma specimen (IgM, 128 mg/dl; IgG, 18 mg/dl; IgE, 1 IU/ml; IgA, 4 mg/dl). The relative proportions of immune cells were CD3 24.6%, CD4 10.3%, CD8 2.2%, CD19 30.2%, CD57 1.0% and active T cells 1.1%. After IVIG treatment, the pneumonia improved. Repeat assessment at the age of 15 months showed IgM decreased to the normal range (32 mg/dl). Whole blood flow cytometry assay for CD40L expression confirmed the diagnosis of hyper-lgM syndrome when he was 21 months old. Only a small percentage (0.48%) of the patient's in vitro activated CD4+ T cells expressed CD40L, compared with 33.54% from a healthy control. The patient's father, mother and sister all had a normal CD40L expression activation patterns (43.52%, 40.78%, 34.11%, respectively). On a regimen of monthly IVIG infusion and oral trimethoprim-sulfamethoxazole for Pneumocystis carinii pneumonia (PCP) prophylaxis, the patient has had no recurrent infections.

Increases in circulating levels of monocyte–platelet and neutrophil–platelet complexes following hip arthroplasty

Platelets and leucocytes are important effector cells of the haemostatic and inflammatory responses to tissue injury. To investigate the effects of surgical trauma on platelet activation (assessed by measuring levels of P-selectin and beta-thromboglobulin), leucocyte activation (CD11b expression) and leucocyte-platelet interactions (leucocyte-platelet complexes), 30 patients undergoing primary hip arthroplasty were studied before and at the end of surgery, and on days 1 and 10 post-operatively, using a whole-blood flow cytometry assay. The inflammatory response was followed by measurement of the levels of C-reactive protein and interleukin-6 in plasma, and the activation of coagulation was monitored by determination of prothrombin fragment 1+2 levels. On day 1 post-operatively a significantly increased expression of CD11b on monocytes was noted, but no direct correlation was found between monocyte activation and interleukin-6 production or C-reactive protein at this time point. The percentage of monocyte-platelet and neutrophil-platelet complexes was markedly increased on day 10 post-operatively compared with pre-operative levels, and levels of these complexes were significantly positively correlated with beta-thromboglobulin levels. Activation of coagulation (prothrombin fragment 1+2) on day 10 post-operatively was positively correlated with the extent of surgical trauma (duration of surgery, amount of blood loss) and with the increase in platelet activation (beta-thromboglobulin). In conclusion, hip arthroplasty induces platelet and coagulation activation, and also an inflammatory response that is maintained for more than 10 days post-operatively. This indicates an interaction between the immune and the haemostatic systems in the post-operative phase after hip arthroplasty.