Whole Blood Flow Cytometry Research Articles

P007 Neutrophil function in lesional biopsies is a strong determinant of IBD severity and may relate to an altered functional state already present in circulating cells

Abstract Background Neutrophils clear luminal bacteria from the intestinal lamina propria and have a protective role in Inflammatory Bowel Disease (IBD). However, their production of inflammatory cytokines, matrix metalloproteases and radical oxygen species causes irreversible tissue damage. Histological assessment of infiltrating neutrophils and indirect measurement of neutrophil-derived calprotectin in feces are used to assess disease severity in patients. Since IBD is a heterogeneous disease in terms of presentation, severity and course, we hypothesize that different patients have a different composition of their neutrophil compartment that leads to a different balance between protection and damage. We aim to study the variability in neutrophil phenotype and function and relate it to patient heterogeneity. Methods We analyzed endoscopic biopsies and peripheral blood samples from children diagnosed with IBD within the PIBD-SETQ cohort. Paired biopsies obtained at diagnosis were scored by a pathologist and analyzed by IHC and RNAseq. RNAseq was also performed on purified circulatory neutrophil at diagnosis and their phenotype was analyzed by whole-blood flow cytometry. Results At diagnosis, high numbers of infiltrating neutrophils in intestinal tissue differentiated IBD patients with high clinical disease activity, more severe endoscopic and histological disease and high plasma concentrations of IL-17. RNA sequencing of lesional intestinal biopsies revealed differential expression of genes linked to neutrophil degranulation and IL-17 signaling in the colon of IBD patients with high neutrophil infiltration. Moreover, these biopsies had high mRNA expression for Oncostatin M, a neutrophil product known to associate with therapy resistance. To assess whether circulating neutrophils were altered in IBD before entering the intestinal tissue, we performed bulk RNAseq analysis of purified circulating neutrophils from non-IBD and CD patients. Neutrophils from CD patients had increased expression of genes related to granule formation and filling (MMP9, S100A8, S100A9) and pathogen sensing (TLR5, NLRC4) but not cytokine production (IL1B, OSM), relative to non-IBD patients. Expression of CD10 was decreased in circulating neutrophils from UC and CD patients while CD16 expression was lower in UC-derived neutrophils, suggesting these cells have a more immature state. Conclusion We have detected changes in the maturity of neutrophils in the circulation of some IBD patients that could have functional consequences in terms of bacterial clearance and tissue damage in the intestinal mucosa. Uncovering different functional profiles in neutrophils may help dissect patient heterogeneity and provide novel targets for therapy.

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.

Inflammatory Response to Intravenously Administered Lipopolysaccharide (LPS) in the Bone Marrow of Healthy Volunteers As a Model for Early Phase Drug Development

Introduction Inflammatory responses in the peripheral and central hematological compartments are important for identification of new biomarkers and drug targets in early phase drug development. There is an increasing interest to investigate the modulation of the inflammatory response upon experimental drug exposure using cellular and molecular analyses, specifically in the central compartment. The lipopolysaccharide (LPS) challenge model allows to study TLR4-mediated local and systemic inflammation and therefore the effects of anti-inflammatory compounds. In addition, there is an interest in the effects of TLR4 agonists on early immune cell populations. The aim of the current study is to investigate the effects of intravenous (i.v.) LPS on the peripheral blood and central bone marrow compartment and to perform a proof-of-methodology study for bone marrow analysis in early phase drug research in healthy volunteers. Methods This was an open-label study in 13 healthy young male subjects, aged 18 - 35 years (inclusive). The study was conducted at the Centre for Human Drug Research, Leiden, The Netherlands. The study protocol was approved by the Medical Ethics Committee. All subjects provided written informed consent prior to any study activity. Subjects were divided into control group (n=3), 1 ng/kg i.v. LPS group (n=5) and 2 ng/kg i.v. LPS group (n=5). Blood and bone marrow samples were collected at baseline and at 4h post-dose timepoints. Primary objective was to evaluate the safety and tolerability of bone marrow sampling in healthy volunteers, by continuous safety monitoring, visual analogue scale (VAS) for pain and subject experience questionnaires. To investigate the pharmacodynamic (PD) effects of i.v. LPS challenge on the bone marrow and whole-blood immune compartments, flow cytometry, cytokine analysis and RNA sequencing was performed. Results All 13 subjects completed the study. Adverse events were mild and reversible, and included puncture site pain related to bone marrow puncture, and flu-like symptoms related to LPS administration. Twelve subjects (92%) indicated they would undergo the procedure again in the future. There was an observable difference in VAS pain score between the baseline bone marrow puncture and LPS treatment groups, but no difference between 1 ng/kg and 2 ng/kg groups. Phenotypic changes of inflammatory signatures in peripheral blood and bone marrow cell populations upon LPS administration were detected by multi-parameter flow cytometry. An LPS-induced inflammatory cytokine profile was present. RNA sequencing revealed differential gene expression in inflammatory gene signatures between peripheral blood mononuclear cells and bone marrow cells. Conclusion We demonstrated that bone marrow analysis is feasible and well-tolerable in early phase studies with healthy volunteers and can be used to assess inflammatory responses in the central hematological compartment upon systemic LPS challenge. Access to bone marrow can provide important novel insights into drug effects on the hematopoiesis and differentiation of immune cells and provides added value to early drug development.

Stability Assessment of Fresh or Cryopreserved Whole Blood and Bone Marrow Samples from AML and B-ALL Patients to Monitor CD123 Receptor and Immune Cells Subsets

Introduction: Compounds targeting CD123 are currently under clinical development for the treatment of acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). As part of clinical trials, CD123 expression and immune cell subsets need be monitored by quantitative flow cytometry (qFC) in bone marrow (BM) aspirates and whole blood (WB) samples before and on-treatment. One critical parameter for analysis is the poor stability of the fresh biological BM or WB samples. No general rule can predict the stability of the expression of a given receptor on diseased or healthy cells thus the pre-analytical stability of each biomarker should be evaluated to design the sample logistics. The pre analytical stability of clinical samples is the main constraint for quantitative flow cytometry (qFC) in multicenter and worldwide clinical studies. The transportation of the samples is one of the limiting factor. The classical freezing method of peripherical whole blood (WB) or bone marrow (BM) isolated by Ficoll requires high skilled operators, is time consuming and may be difficult to set up at sampling site in hospitals. One option to make the clinical samples suitable for subsequent high dimensional analysis by qFC is to use a simple freezing method at -80°C. Our cryopreservation method could be a new option for easier preparation of clinical samples prior to centralized samples analysis. This study aims to evaluate the pre-analytical fresh or cryopreserved stability of each pharmacodynamic monitoring endpoint over time: CD123 expression and receptor density, T-cell subsets and activation profile. Methods: FreshBM and WB were collected in EDTA tubes from 5 AML/MDS patients at hospitals, in accordance with local regulations. The samples were stored at room temperature (RT) for 4, 24, 48 or 72 h before immunostaining with the standard operation procedures and analysis by qFC or mixed with the cryopreserved media (BioCytex) within 24 h post-collection before freezing at -80°C for months. After storage, the cryopreserved samples were thawed and immunostained before analysis by qFC. Using a qFC assay (Multiplex FC panel assay developed by BioCytex, Marseille, France), CD123 receptor density/cell was collected. T-cell and blast cell immunophenotypes were also assessed on fresh samples for time point 4, 24, 48, or 72 h post-collection but also for cryopreserved WB sample within the 24hs post collection. The assay profiled blasts, monocytes, plasmacytoid dendritic cells, basophils and T-cells. Results: CD123 density in blast and normal cells was stable at RT from 4 h up to 48 h after collection. The profiles of blast subsets were very similar in BM and WB samples from a particular patient, whether a single blast cell subset (CD45lowCD11b-CD34+CD38med) or several blast cell subsets (expressing CD34 and CD38 at different levels) were identified. Both T-cell distribution and activation markers were stable up to 48 h for BM and WB. A similar pattern of T-cell subsets was also observed in the BM and WB paired samples from the same donor. In addition, CD123 receptor density, immunophenotyping and percentage of blast subsets and T cells from fresh WB samples was comparable to paired cryopreserved samples with a new method and for at least 1 month. Conclusions: Based on these observations, we conclude that CD123 density, blast cells and T-cell subsets in WB and BM samples from AML patients were stable for 48 h in our experimental conditions. As a consequence, fresh WB and BM samples can be processed and analyzed by qFC within 48 h of sample collection. Furthermore, the new cryopreservation method showed sample stabilization for blasts and T cells immunophenotyping. This data opens new perspectives in clinical studies for AML This new strategy would also help to standardize preparation of clinical samples and to centralize sample analysis.

The role of TLR2 in exercise-induced immunomodulation in normal weight individuals

Toll-like receptors (TLRs) have been targeted for therapeutic drug development for several disorders, including cardiovascular diseases (CVD), and diabetes mellitus. Daily levels physical activity (PA) has been purported to influence the systemic circulation of cytokines, affecting the overall activation of TLRs and influencing the inflammatory milieu. Objective and self-reported daily PA was tracked in 69 normal-weight adults. Freedson's cut-offs categorized daily PA intensity into the 25th lowest, medium, and top percentiles. Monocytic TLR2 expression was quantified by flow cytometry in fresh whole blood. Cross-sectional associations between flow cytometry measured TLR2+ subsets and clinical biomarkers were evaluated. PA increased circulation of TLR2+ monocytes. TLR2 expression was adversely corelated with reduced diastolic blood pressure (DBP), triglyceride (TG), and matrix metallopeptidase 9 (MMP9) levels. However, regression analysis indicated that only TG levels were independently linked with TLR2+ subsets in circulation in active participants. Higher daily levels of physical activity are associated with improved cardiovascular blood markers and elevated circulatory monocytic TLR2+ subsets. These findings suggest that TLR2 may play a role in modulating CVD risk factors in individuals leading physically active lifestyles.

Third SARS-CoV-2 vaccination and breakthrough infections enhance humoral and cellular immunity against variants of concern.

SARS-CoV-2 vaccination is the leading strategy to prevent severe courses after SARS-CoV-2 infection. In our study, we analyzed humoral and cellular immune responses in detail to three consecutive homologous or heterologous SARS-CoV-2 vaccinations and breakthrough infections. Peripheral blood samples of n=20 individuals were analyzed in the time course of three SARS-CoV-2 vaccinations and/or breakthrough infection. S1-, RBD-, S2- and N-specific IgG antibodies were quantified using Luminex-based multiplex assays and electrochemiluminescence multiplex assays for surrogate neutralization in plasma. Changes in cellular immune components were determined via flow cytometry of whole blood samples. All individuals (n=20) responded to vaccination with increasing S1-/RBD-/S2-specific IgG levels, whereas specific plasma IgA displayed individual variability. The third dose increased antibody inhibitory capacity (AIC) against immune-escape variants Beta and Omicron BA.1 independently of age. The mRNA-primed vaccination induced IgG and IgA immunity more efficiently, whereas vector-primed individuals displayed higher levels of memory T and B cells. Vaccinees showed SARS-CoV-2-specific T cell responses, which were further improved and specified after Omicron breakthrough infections in parallel to the appearance of new variant-specific antibodies. In conclusion, the third vaccination was essential to increase IgG levels, mandatory to boost AIC against immune-escape variants, and induced SARS-CoV-2-specific T cells. Breakthrough infection with Omicron generates additional spike specificities covering all known variants.

Effect of Hydrogen Sulfide on Essential Functions of Polymorphonuclear Leukocytes.

Impaired polymorphonuclear leukocyte (PMNL) functions contribute to increased infections and cardiovascular diseases in chronic kidney disease (CKD). Uremic toxins reduce hydrogen sulfide (H2S) levels and the anti-oxidant and anti-inflammatory effects of H2S. Its biosynthesis occurs as a side process of transsulfuration and in the disposal of adenosylhomocysteine, a transmethylation inhibitor and proposed uremic toxin. PMNL chemotaxis was measured by the under-agarose method, phagocytosis, and oxidative burst by flow cytometry in whole blood and apoptosis by determining DNA content by flow cytometry and morphological features by fluorescence microscopy. Sodium hydrogen sulfide (NaHS), diallyl trisulphide (DATS) and diallyl disulphide (DADS), cysteine, and GYY4137 were used as H2S-producing substances. Increased H2S concentrations did not affect chemotaxis and phagocytosis. NaHS primed PMNL oxidative burst activated by phorbol 12-myristate 13-acetate (PMA) or E. coli. Both DATS and cysteine significantly decreased E. coli-activated oxidative burst but had no effect on PMA stimulation. While NaHS, DADS, and cysteine attenuated PMNL apoptosis, GYY4137 decreased their viability. Experiments with signal transduction inhibitors suggest that the intrinsic apoptosis pathway is mainly involved in GYY4137-induced PMNL apoptosis and that GYY4137 and cysteine target signaling downstream of phosphoinositide 3-kinase.

Early SARS-CoV-2 infection: Platelet-neutrophil complexes and platelet function

BackgroundConflicting results have been reported on platelet activity ex vivo and responsiveness in vitro among patients with COVID-19 with or without thromboembolic complications. ObjectivesTo assess platelet reactivity in patients with moderate disease at early stages of COVID-19. MethodsWe performed a prospective, descriptive analysis of 100 consecutive patients presenting with suspected SARS-CoV-2 infection at University Medical Center Freiburg during the first or second wave of the pandemic. Following polymerase chain reaction testing and compliance with study inclusion criteria, 20 SARS-CoV-2–positive and 55 SARS-CoV-2–negative patients (serving as patient controls) were enrolled. In addition, 15 healthy subjects were included. Platelet reactivity was assessed using whole-blood impedance aggregometry and flow cytometry in response to various agonists. ResultsPlatelet aggregation was significantly impaired in the patients with COVID-19 compared with that in the patient controls or healthy subjects. The reduced platelet responsiveness in the patients with COVID-19 was associated with impaired activation of GPIIb/IIIa (αIIbβ3). In contrast, low expression of P-selectin at baseline and intact secretion upon stimulation in vitro suggest that no preactivation in vivo, leading to “exhausted” platelets, had occurred. The proportion of circulating platelet-neutrophil complexes was significantly higher in the patients with COVID-19 (mean ± SD, 41% ± 13%) than in the patient controls (18% ± 7%; 95% CI, 11.1-34.1; P = .0002) or healthy subjects (17% ± 4%; 95% CI, 13.8-33.8; P < .0001). An analysis of neutrophil adhesion receptors revealed upregulation of CD11b (α-subunit of αMβ2) and CD66b (CEACAM8) but not of CD162 (PSGL-1) in the patients with COVID-19. ConclusionDespite reduced platelet responsiveness, platelet-neutrophil complexes are increased at early stages of moderate disease. Thus, this cellular interaction may occur during COVID-19 without preceding platelet activation.

Altered compositions of monocyte, T lymphocyte and NK cell subsets in heart failure of adult congenital heart disease

BackgroundAdults with congenital heart disease (ACHD) display changes in adaptive immunity related to early open-heart surgery and subsequent incidental thymectomy. In acquired heart failure (HF) systemic inflammation, innate and adaptive immune cells play an important role. However, cellular immune alterations of monocyte, T lymphocyte and natural killer (NK) cell subsets have not been related to HF in ACHD. Methods and resultsThis cross-sectional study included 209 ACHD outpatients (mean age: 35.3 ± 11.0 years; NYHA class I/II/III-IV 64.1%/21.5%/14.4%; 59.8% male) and 21 healthy controls (29.8 ± 12.6 years; 47.6% male). Patients with clinical signs of infection, inflammatory diseases or malignancies were excluded. Flow cytometry of fresh whole blood revealed significantly elevated levels of CD14++CD16+ (Mon2) and CD14+CD16++ (Mon3) monocytes that increased in advanced NYHA class. Immature CD14+HLA-DRneg/low monocytes were significantly higher in ACHD, but did not relate to NT-proBNP or NYHA class. Frequencies of Mon2, CD14+HLA-DRneg/low and neutrophils related to plasma S100A8/A9 levels. Furthermore, ACHD had decreased T helper and cytotoxic T cell counts, however NKT and CD3bright T cells remained unchanged compared to control. The cytokine-producing CD56bright NK subset related to NT-proBNP and expanded in advanced HF stages. Patients with elevated Mon2 and CD14+HLA-DRneg/low monocytes exhibited a worse outcome regarding all-cause mortality/cardiac decompensations after a mean follow-up of 5.5 years. High Mon2, low total lymphocyte and low T helper cell counts were independent prognostic markers for the onset of adverse cardiac events. ConclusionsAltered immune cell compositions related to heart failure and exhibited prognostic relevance. This suggests an important link between immunity, inflammation and HF in ACHD bearing potential for refined clinical risk stratification.

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.

SARS-CoV-2 Omicron BA.1 variant breakthrough infections in nursing home residents after an homologous third dose of the Comirnaty® COVID-19 vaccine: Looking for correlates of protection.

We investigated whether peripheral blood levels of SARS‐CoV‐2 Spike (S) receptor binding domain antibodies (anti‐RBD), neutralizing antibodies (NtAb) targeting Omicron S, and S‐reactive‐interferon (IFN)‐γ‐producing CD4+ and CD8+ T cells measured after a homologous booster dose (3D) with the Comirnaty® vaccine was associated with the likelihood of subsequent breakthrough infections due to the Omicron variant. An observational study including 146 nursing home residents (median age, 80 years; range, 66–99; 109 female) evaluated for an immunological response after 3D (at a median of 16 days). Anti‐RBD total antibodies were measured by chemiluminescent immunoassay. NtAb were quantified by an Omicron S pseudotyped virus neutralization assay. SARS‐CoV‐2‐S specific‐IFNγ‐producing CD4+ and CD8+ T cells were enumerated by whole‐blood flow cytometry for intracellular cytokine staining. In total, 33/146 participants contracted breakthrough Omicron infection (symptomatic in 30/33) within 4 months after 3D. Anti‐RBD antibody levels were comparable in infected and uninfected participants (21 123 vs. 24 723 BAU/ml; p = 0.34). Likewise, NtAb titers (reciprocal IC50 titer, 157 vs. 95; p = 0.32) and frequency of virus‐reactive CD4+ (p = 0.82) and CD8+ (p = 0.91) T cells were similar across participants in both groups. anti‐RBD antibody levels and NtAb titers estimated at around the time of infection were also comparable (3445 vs. 4345 BAU/ml; p = 0.59 and 188.5 vs. 88.9; p = 0.70, respectively). Having detectable NtAb against Omicron or SARS‐CoV‐2‐S‐reactive‐IFNγ‐producing CD4+ or CD8+ T cells after 3D was not correlated with increased protection from breakthrough infection (OR, 1.50; p = 0.54; OR, 0.0; p = 0.99 and OR 3.70; p = 0.23, respectively). None of the immune parameters evaluated herein, including NtAb titers against the Omicron variant, may reliably predict at the individual level the risk of contracting COVID‐19 due to the Omicron variant in nursing home residents.

Immunological correlates of response and immune-mediated toxicity in checkpoint inhibitor (ICI)-treated metastatic urothelial carcinoma (mUC) patients (pts).

526 Background: Myeloid derived suppressors cells (MDSC) are immune cells that create an immunosuppressive microenvironment. Increased expression of MDSC subsets is associated with worse overall survival in ICI-treated mUC pts, but their role in immune-related adverse events (irAE) is unknown. Immune profiles associated with irAE are also unknown. We investigated associations of MDSC and –omics profiles with response and irAE in ICI-treated mUC pts. Methods: Baseline (B) and on-treatment (Tx) blood samples were collected from ICI-treated mUC pts. MDSC were measured in fresh unfractionated whole blood (WB) and in peripheral blood mononuclear cells (PBMC). MDSC were identified by flow cytometry in WB, defined as LinloCD33+/HLADR-, and subclassified as polymorphonuclear (PMN)-MDSC (CD15+/CD14-), monocytic (M)-MDSC (CD15-/CD14+), and uncommitted (UC)-MDSC (CD15-/CD14-). MDSC populations were presented as % of live nucleated blood cells and as absolute numbers from WB. irAE severity was graded by CTCAE v5. In a subcohort of 17 pts, proteomics and transcriptomics were analyzed via Olink and Bulk RNAseq, respectively. Wilcoxon rank sum test compared MDSC and –omics among response and irAE groups. Kruskal-Wallis test compared –omics results between irAE responders (irAE-R), irAE non-responders (irAE-NR), and no irAE/non-responders (noAE-NR). Results: 41 ICI-treated mUC pts (25 anti-PD-L1, 16 anti-PD-1) had at least 1 MDSC sample: 28 pts at B, 30 pts at Tx, and 17 pts at both B and Tx. Primary UC sites were bladder (78%) and upper tract (22%); 73% male; median age 72 (range, 28-82); 85% had KPS &gt; 80%; 51% had visceral metastasis. ICI was first and second-line therapy in 37% and 63% of pts, respectively. 13 pts were responders (R); 26 pts were non-responders (NR); 2 pts were not evaluable. 22 pts developed irAE. Median time to irAE was 84 days (range, 21-145); 10 pts required steroids; 3 required ICI discontinuation. UC-MDSC was predominant in WB and PMN-MDSC in PBMC in both B and Tx. Between B and Tx, WB UC-MDSC and PB UC-MDSC increased in R (n = 13; p = 0.04), but decreased in NR (n = 26; p = 0.02). In the subcohort of 17 pts, 11 had irAE (7 irAE-R; 4 irAE-NR), 6 had noAE-NR. Proteomic analysis showed increased expression of CXCL12 in noAE-NR pts (p = 0.006) and increased expression of IL-8 (p = 0.016), IL-18 (p = 0.012), and IL-18R1 (p = 0.016) in all irAE pts. At the transcriptome level, upregulation of IFN-γ was associated with response, whereas upregulation of both IFN-γ and IFN-α differentiated irAE-R from irAE-NR. Conclusions: In ICI-treated mUC pts, WB &amp; PB UC-MDSC increased in R and decreased in NR between B and Tx. Increased expression of pro-inflammatory chemokines was observed in irAE pts, independent of response. A distinct inflammatory pathway was observed in irAE-R. Prospective investigation of blood-based biomarkers of response and irAE development is warranted.

Normal ex vivo mesenchymal stem cell function combined with abnormal immune profiles sets the stage for informative cell therapy trials in idiopathic pulmonary fibrosis patients

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease characterized by aberrant tissue remodeling, formation of scar tissue within the lungs and continuous loss of lung function. The areas of fibrosis seen in lungs of IPF patients share many features with normal aging lung including cellular senescence. The contribution of the immune system to the etiology of IPF remains poorly understood. Evidence obtained from animal models and human studies suggests that innate and adaptive immune processes can orchestrate existing fibrotic responses. Currently, there is only modest effective pharmacotherapy for IPF. Mesenchymal stem cells (MSCs)-based therapies have emerged as a potential option treatment of IPF. This study characterizes the functionality of autologous MSCs for use as an IPF therapy and presents an attempt to determine whether the disease occurring in the lungs is associated with an alterated immune system.MethodsComprehensive characterization of autologous adipose-derived MSCs (aMSCs) from 5 IPF patient and 5 age- and gender-matched healthy controls (HC) was done using flow cytometry, PCR (ddPCR), multiplex Luminex xMAP technology, confocal microscopy self-renewal capacity and osteogenic differentiation. Additionally, multi-parameter quantitative flow cytometry of unmanipulated whole blood of 15 IPF patients and 87 (30 age- and gender-matched) HC was used to analyze 110 peripheral phenotypes to determine disease-associated changes in the immune system.ResultsThere are no differences between autologous aMSCs from IPF patients and HC in their stem cell properties, self-renewal capacity, osteogenic differentiation, secretome content, cell cycle inhibitor marker levels and mitochondrial health. IPF patients had altered peripheral blood immunophenotype including reduced B cells subsets, increased T cell subsets and increased granulocytes demonstrating disease-associated alterations in the immune system.ConclusionsOur results indicate that there are no differences in aMSC properties from IPF patients and HC, suggesting that autologous aMSCs may be an acceptable option for IPF therapy. The altered immune system of IPF patients may be a valuable biomarker for disease burden and monitoring therapeutic response,

Evolution of platelet activation parameters during septic shock in intensive care unit

During severe sepsis, platelet activation may induce disseminate microvascular thrombosis, which play a key role in critical organ failure. Crucially, most of the studies in this field have explored platelet–leukocyte interactions in animal models, or explored platelets under the spectrum of thrombocytopenia or disseminated intravascular coagulation and have not taken into account the complex interplay that might exist between platelets and leukocytes during human septic shock nor the kinetics of platelet activation. Here, we assessed platelet activation parameters at the admission of patients with sepsis to the intensive care unit (ICU) and 48 hours later. Twenty-two patients were enrolled in the study, thirteen (59.1%) of whom were thrombocytopenic. The control group was composed of twelve infection-free patients admitted during the study period. The activation parameters studied included platelet-leukocyte interactions, assessed by flow cytometry in whole blood, as well as membrane surface and soluble platelet activation markers measured by flow cytometry and dedicated ELISA kits. We also investigated platelet aggregation and secretion responses of patients with sepsis following stimulation, compared to controls. At admission, the level of circulating monocyte-platelet and neutrophil-platelet heterotypic aggregates was significantly higher in sepsis patients compared to controls and returned to a level comparable to controls or even below 48 hours later. Basal levels of CD62P and CD63 platelet membrane exposure at admission and 48 hours later were low and similar to controls. In contrast, plasma level of soluble GPVI and soluble CD40 ligand was significantly increased in septic patients, at the two times of analysis, reflecting previous platelet activation. Platelet aggregation and secretion responses induced by specific agonists were significantly decreased in septic conditions, particularly 48 hours after admission. Hence, we have observed for the first time that critically ill septic patients compared to controls have both an early and durable platelet activation while their circulating platelets are less responsive to different agonists.

New technologies for diagnosing active TB: the VANTDET diagnostic accuracy study

Background Tuberculosis (TB) is a devastating disease for which new diagnostic tests are desperately needed. Objective To validate promising new technologies [namely whole-blood transcriptomics, proteomics, flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)] and existing signatures for the detection of active TB in samples obtained from individuals with suspected active TB. Design Four substudies, each of which used samples from the biobank collected as part of the interferon gamma release assay (IGRA) in the Diagnostic Evaluation of Active TB study, which was a prospective cohort of patients recruited with suspected TB. Setting Secondary care. Participants Adults aged ≥ 16 years presenting as inpatients or outpatients at 12 NHS hospital trusts in London, Slough, Oxford, Leicester and Birmingham, with suspected active TB. Interventions New tests using genome-wide gene expression microarray (transcriptomics), surface-enhanced laser desorption ionisation time-of-flight mass spectrometry/liquid chromatography–mass spectrometry (proteomics), flow cytometry or qRT-PCR. Main outcome measures Area under the curve (AUC), sensitivity and specificity were calculated to determine diagnostic accuracy. Positive and negative predictive values were calculated in some cases. A decision tree model was developed to calculate the incremental costs and quality-adjusted life-years of changing from current practice to using the novels tests. Results The project, and four substudies that assessed the previously published signatures, measured each of the new technologies and performed a health economic analysis in which the best-performing tests were evaluated for cost-effectiveness. The diagnostic accuracy of the transcriptomic tests ranged from an AUC of 0.81 to 0.84 for detecting all TB in our cohort. The performance for detecting culture-confirmed TB or pulmonary TB was better than for highly probable TB or extrapulmonary tuberculosis (EPTB), but was not high enough to be clinically useful. None of the previously described serum proteomic signatures for active TB provided good diagnostic accuracy, nor did the candidate rule-out tests. Four out of six previously described cellular immune signatures provided a reasonable level of diagnostic accuracy (AUC = 0.78–0.92) for discriminating all TB from those with other disease and latent TB infection in human immunodeficiency virus-negative TB suspects. Two of these assays may be useful in the IGRA-positive population and can provide high positive predictive value. None of the new tests for TB can be considered cost-effective. Limitations The diagnostic performance of new tests among the HIV-positive population was either underpowered or not sufficiently achieved in each substudy. Conclusions Overall, the diagnostic performance of all previously identified ‘signatures’ of TB was lower than previously reported. This probably reflects the nature of the cohort we used, which includes the harder to diagnose groups, such as culture-unconfirmed TB or EPTB, which were under-represented in previous cohorts. Future work We are yet to achieve our secondary objective of deriving novel signatures of TB using our data sets. This was beyond the scope of this report. We recommend that future studies using these technologies target specific subtypes of TB, specifically those groups for which new diagnostic tests are required. Funding This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a MRC and NIHR partnership.

JAK selectivity and the implications for clinical inhibition of pharmacodynamic cytokine signalling by filgotinib, upadacitinib, tofacitinib and baricitinib

ObjectiveJanus kinase inhibitors (JAKinibs) are efficacious in rheumatoid arthritis (RA) with variable reported rates of adverse events, potentially related to differential JAK family member selectivity. Filgotinib was compared with baricitinib,...

Relationship Between Composition of Fatty Acid in Platelet Phospholipid Membrane and Markers of Oxidative Stress in Healthy Men and Men After a Myocardial Infarction.

BackgroundOxidative stress (OS) is known to be extremely damaging for phospholipids in cell membranes, especially their polyunsaturated fatty acids (PUFAs). OS is known to be associated with increased platelet activation and thrombosis, which lead to cardiovascular lesions. The aim of this study was to investigate how changes in the composition of fatty acids (FAs) in the platelet phospholipid membrane correlate with OS in healthy men and in men who have experienced a myocardial infarction (post-MI men).Material/MethodsFA methyl esters from the platelet phospholipid membrane of 79 apparently healthy and 20 post-MI men were identified using gas chromatography/mass spectrometry. Malondialdehyde (MDA) was measured in the blood serum using high-performance liquid chromatography, and platelet-white blood cell aggregates (PWAs) were analysed based on whole-blood flow cytometry. The composition of platelet membrane FAs was compared to MDA concentration (μg/l) and the percentage of PWA formation between healthy men and individuals who had suffered a myocardial infarction (MI).ResultsStatistically, post-MI patients had a significantly higher concentration of blood serum MDA than those in the control group (p=0.000). The level of PUFAs was also higher in the platelet phospholipid membrane of post-MI patients than in healthy individuals (p=0.016). However, the percentage of PWA formation was lower in patients compared with the control group (p<0.05).ConclusionsA higher level of blood serum MDA concentration due to OS stimulates platelets to incorporate more PUFAs into the phospholipid membrane, thereby affecting platelet activation. This may lead the individual to develop cardiovascular diseases in the future.

Preliminary Evaluation of an Autologous Dendritic Cell Vaccine Using Nanoparticle Technology for the Treatment of Canine Malignant Melanoma

Canine malignant melanoma is an aggressive neoplasm that carries a poor prognosis due to its minimal responsiveness to traditional therapy protocols, particularly if the oral cavity, mucocutaneous junctions, or subungual sites are involved. This proof-of-concept study evaluated a prototype autologous dendritic cell vaccine using poly-lactic-co-glycolic (PLGA) nanoparticles containing antigens from patient-derived whole tumor lysate and the adjuvant monophosphoryl lipid A in five canines with stages III-IV malignant melanoma. Nanoparticle constructs biochemical characterization; encapsulation efficiency and kinetic release studies were determined. Our results showed that tumor antigens were successfully incorporated in the PLGA/monophosphoryl lipid A nanoparticle constructs. Additional in vitro experiments showed that the PLGA/monophosphoryl lipid A nanoparticle constructs effectively activated autologous dendritic cells, and generated a greater than twofold increase in the release of the pro-immune cytokine IFN-γ. No significant adverse effects were observed in any of the patients following intradermal vaccination, and flow cytometry of whole blood revealed increased CD4:CD8 T lymphocyte ratios by the completion of the study. These results suggest that a dendritic cell vaccine utilizing PLGA/monophosphoryl lipid A nanoparticle technology could potentially initiate an adaptive immune response and is safe to administer to canine patients. Further in vivo studies with a larger cohort of patients are warranted.

Circulating Regulatory T-Cell Number Does Not Predict Prognosis of Monoclonal Gammopathies of Uncertain Significance.

FOXP3-expressing regulatory T-cells (Tregs), which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. It has been shown that there is an increased proportion of Tregs in several different human malignancies, although the actual mechanism remains unclear. The research aims to explore the relationship between the number of Tregs and a predict prognosis in particular hematological diseases as monoclonal gammopathies of uncertain significance (MGUS). Tregs were evaluated by means of flow cytometry (CD4+CD25high/+ CD127low/-) in whole peripheral blood of 56 patients with MGUS to predict progression to overt multiple myeloma (MM). In two groups of patients, MGUS versus MGUS evolved to MM, we found a significative difference for the number of white blood cells, but not in terms of clinical and laboratory features evaluated at diagnosis. The study demonstrated the absence of a prognostic relevance of Tregs in MGUS. Nevertheless, their role in these disorders is still to be defined.

Features of peripheral blood B-cell subset phenotype are associated with clinical outcome of widespread purulent peritonitis

The aim of the study was to investigate the phenotypic features of peripheral blood B-lymphocytes in patients with widespread purulent peritonitis (WPP) during postoperative treatment depending on the disease outcome. 52 patients with acute surgical diseases and injuries of the abdominal organs complicated by WPP were examined. Blood sampling was performed before surgery (preoperative period), as well as on day 7, 14 and 21 of the postoperative period. All patients with WPP were divided into two groups depending on the disease outcome during postoperative period: patients with favorable outcome (n = 34), patients with unfavorable outcome (n = 18). 68 healthy subjects were included into control group. Immunophenotyping of blood B-lymphocytes was assessed by flow cytometry and direct immunofluorescence of whole peripheral blood stained with monoclonal antibodies. It was found that patients with WPP in preoperative period contained lowered B1-cell subset compared to control group that was paralleled with low absolute B-lymphocyte count. Moreover, a higher number of activated (based on upregulated CD23 expression) peripheral blood B1-lymphocytes was observed in the patients with unfavorable than in patients with favorable WPP outcome. In addition, dynamics of changes in frequency of B-lymphocytes during postoperative period (day 7—21) varied profoundly depending on the disease outcome. In particular, patients with favorable WPP outcome during postoperative period were found to contain decreased percentage of the most B-cell subsets (including activated cells) examined, whereas patients with unfavorable disease outcome had virtually unaltered B-cell composition in the postoperative period. Moreover, count of total B-cells, naive B-cells and B2-cells negative or positive for CD23 expression was higher in patients with unfavorable outcome than in patients with favorable WPP outcome throughout entire postoperative period. Percentage of some other B-cell subsets in patients with unfavorable than with favorable outcome was also higher only at certain stages of postoperative treatment. It is assumed that such features in peripheral blood B-cell subset composition were closely linked to the disease outcome so that patients with unfavorable WPP outcome turned out to have disturbed B-cell maturation and migration in developing immune response that might be due to lowered total sensitivity of host body to postoperative antibiotic therapy.