Peripheral Blood Mononuclear Cell Subsets Research Articles

Expression of Sex Hormone Receptor and Immune Response Genes in Peripheral Blood Mononuclear Cells During the Menstrual Cycle.

Sex hormones are known to interact with the immune system on multiple levels but information on the types of sex hormone receptors (SHR) and their expression levels in immune cells is scarce. Estrogen, testosterone and progesterone are all considered to interact with the immune system through their respective cell receptors (ERα and ERβ including the splice variant ERβ2, AR and PGR). In this study expression levels of SHR genes in peripheral blood mononuclear cells (PBMCs) and cell subsets (CD4+ and CD8+ T-cells, CD56+ NK-cells, CD14+ monocytes and CD19+ B-cells) were analyzed using standard manual qPCR or a qPCR array (TLDA). Nine healthy individuals including men (n = 2), premenopausal (Pre-MP, n = 5) and postmenopausal (post-MP, n = 2) women were sampled for PBMCs which were separated to cell subsets using FACS. Ten Pre-MP women were longitudinally sampled for total PBMCs at different phases of the menstrual cycle. We found that ERα was most abundant and, unexpectedly, that ERβ2 was the dominant ERβ variant in several FACS sorted cell subsets. In total PBMCs, SHR (ERα, ERβ1, ERβ2, and AR) expression did not fluctuate according to the phase of the menstrual cycle and PGR was not expressed. However, several immune response genes (GATA3, IFNG, IL1B, LTA, NFKB1, PDCD1, STAT3, STAT5A, TBX21, TGFB1, TNFA) were more expressed during the ovulatory and mid-luteal phases. Sex hormone levels did not correlate significantly with gene expression of SHR or immune response genes, but sex hormone-binding globulin (SHBG), a steroid hormone transporting protein, was positively correlated to expression of ERβ1 gene. This study provides new insights in the distribution of ERs in immune cells. Furthermore, expression patterns of several immune response genes differ significantly between phases of the menstrual cycle, supporting a role for sex hormones in the immune response.

MR spectroscopy and diffusion imaging in people with human immunodeficiency virus: Relationships to clinical and immunologic findings.

People with human immunodeficiency virus (HIV; PWH) present a complex array of immunologic and medical disorders that impact brain structure and metabolism, complicating the interpretation of neuroimaging. This pilot study of well-characterized multi-morbid PWH examined how medical and immunologic factors predicted brain characteristics on proton MR spectroscopy (1H-MRS) and diffusion-weighted imaging (DWI). Eighteen individuals on combination antiretroviral therapy (cART), with mean age of 56 years, underwent medical history review, neuroimaging, and on the day of imaging, blood draw for assay of 20 plasma cytokines and flow cytometric characterization of peripheral blood mononuclear cell subsets. Predictors of n-acetyl aspartate, choline, myoinositol, glutamate/glutamine, fractional anisotropy and mean diffusivity were identified through bivariate correlation; those significant at p < .1000 were advanced to multivariate analysis, with models created for each neuroimaging outcome. Monocyte subsets and diverse cytokines accounted for 16 of 25 (64%) variables predicting 1H-MRS spectra in frontal gray and white matter and basal ganglia; monocyte subsets did not predict any DWI characteristic. In contrast, age, presence of hypertension, and duration of HIV infection accounted for 13 of 25 (52%) variables predicting diffusion characteristics in the corpus callosum, thalamic radiations, and basal ganglia but only 3 of 25 (12%) predictors of 1H-MRS features. 1H-MRS neurometabolites were most often predicted by immunologic factors sensitive to temporal variation, whereas DWI metrics were more often related to longer-term disease state. In multi-morbid cART-era populations, selection and interpretation of neuroimaging modalities should account for complex temporal and pathogenetic influences of immunologic abnormality, disease state, and aging.

Identifying novel ovarian tumor biomarkers through mining of the transcriptome of circulating immune cells: A proof-of-concept study.

Treatment of high-grade serous ovarian cancer (HGSOC) will benefit from early detection of cancer. Here, we provide proof-of-concept data supporting the hypothesis that circulating immune cells, because of their early recognition of tumors and the tumor microenvironment, can be considered for biomarker discovery. Longitudinal blood samples from C57BL/6 mice bearing syngeneic ovarian tumors and peripheral blood mononuclear cells (PBMC) from healthy postmenopausal women and newly diagnosed for HGSOC patients were subjected to RNASeq. The results from human immune cells were validated using Affymetrix microarrays. Differentially expressed transcripts in immune cells from tumor-bearing mice and HGSOC patients were compared to matching controls. A total of 1282 transcripts (798 and 484, up- and downregulated, respectively) were differentially expressed in the tumor-bearing mice as compared with controls. Top 100 genes showing longitudinal changes in gene expression 2, 4, 7, and 18days after tumor implantation were identified. Analysis of the PBMC from healthy post-menopausal women and HGSOC patients identified 4382 differentially expressed genes and 519 of these were validated through Affymetrix microarray analysis. A total of 384 genes, including IL-1R2, CH3L1, Infitm1, FP42, CXC42, Hdc, Spib, and Sema6b, were differentially expressed in the human and mouse datasets. The PBMC transcriptome shows longitudinal changes in response to the progressing tumor. Several potential biomarker transcripts were identified in HGSOC patients and mouse models. Monitoring their expression in individual PBMC subsets can serve as additional discriminator for the diagnosis of HGSOC.

Abstract 1525: Enhancing potency of antigen presenting cells via signal 2/3 mRNA engineering through Cell Squeeze® technology

Abstract Antigen-specific CD8+ T cells are critical effectors of the immune system that help limit persistence of tumors and virally infected cells. During priming, T cells integrate three signals which determine the magnitude and quality of response generated. Signal one is T cell receptor(TCR) and peptide-MHC (pMHC) engagement, which determines the specificity of the response.Signal two is cell surface costimulation by antigen presenting cells (APCs). Signal three is provided through the local cytokine milieu at the time of priming. Here, we use microfluidic CellSqueeze® technology to deliver mRNAs encoding antigen (signal 1), costimulatory molecules(signal 2), and chimeric membrane-tethered cytokines (signal 3) to the cytosol of human peripheral blood mononuclear cells (PBMCs), generating antigen presenting cells (APCs) with multiple enhanced functions. We demonstrate that microfluidic squeezing enables delivery and expression of single or multiple mRNAs encoding signal 1 (various antigens), signal 2 (CD70 orCD86) and/or signal 3 (membrane-tethered form of IL-2) by the major subsets of PBMCs (T cells, B cells, NK cells, and monocytes). While unsqueezed PBMCs showed no to minimal expression of signal 2 and no expression of signal 3 molecules, expression of delivered signal 2and 3 mRNAs in squeezed PBMCs could be observed on the cell surface for several days post squeeze delivery - a timeframe that could potentially support improved T cell priming. When these signal 2/3 molecules were delivered alone or in combination, antigen-specific CD8+ T cell responses could be increased as much as ten-fold compared to delivery of antigen alone.Therefore, microfluidic cell squeezing enables us to efficiently deliver mRNA antigens that have potential to generate multiple immune epitopes in an HLA agnostic manner. Moreover,multiplexing these antigens with signal 2/3 mRNAs enhances the antigen presenting potency ofSQZ APCs inducing stronger antigen-specific CD8+ T cell responses. The potential to deliver numerous materials simultaneously and engineer compound signals via mRNA could allow for applications for HLA-agnostic patient population in oncology and infectious disease areas. Citation Format: Emrah Ilker Ozay, Paul Dunbar, Kelly Volk, Michael F. Maloney, Christian Yee, Mubeen Mosaheb, Christine Trumpfheller, Pablo Umana, Katherine J. Seidl, Howard Bernstein, Scott M. Loughhead. Enhancing potency of antigen presenting cells via signal 2/3 mRNA engineering through Cell Squeeze® technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1525.

Systems Biology behind Immunoprotection of Both Sheep and Goats after Sungri/96 PPRV Vaccination.

ABSTRACTImmune response is a highly coordinated cascade involving all the subsets of peripheral blood mononuclear cells (PBMCs). In this study, RNA sequencing (RNA-Seq) analysis of PBMC subsets was done to delineate the systems biology behind immune protection of the vaccine in sheep and goats. The PBMC subsets studied were CD4+, CD8+, CD14+, CD21+, and CD335+ cells from day 0 and day 5 of sheep and goats vaccinated with Sungri/96 peste des petits ruminants virus. Assessment of the immune response processes enriched by the differentially expressed genes (DEGs) in all the subsets suggested a strong dysregulation toward the development of early inflammatory microenvironment, which is very much required for differentiation of monocytes to macrophages, and activation as well as the migration of dendritic cells into the draining lymph nodes. The protein-protein interaction networks among the antiviral molecules (IFIT3, ISG15, MX1, MX2, RSAD2, ISG20, IFIT5, and IFIT1) and common DEGs across PBMC subsets in both species identified ISG15 to be a ubiquitous hub that helps in orchestrating antiviral host response against peste des petits ruminants virus (PPRV). IRF7 was found to be the key master regulator activated in most of the subsets in sheep and goats. Most of the pathways were found to be inactivated in B lymphocytes of both the species, indicating that 5 days postvaccination (dpv) is too early a time point for the B lymphocytes to react. The cell-mediated immune response and humoral immune response pathways were found more enriched in goats than in sheep. Although animals from both species survived the challenge, a contrast in pathway activation was observed in CD335+ cells.IMPORTANCE Peste des petits ruminants (PPR) by PPR virus (PPRV) is an World Organisation for Animal Health (OIE)-listed acute, contagious transboundary viral disease of small ruminants. The attenuated Sungri/96 PPRV vaccine used all over India against this PPR provides long-lasting robust innate and adaptive immune response. The early antiviral response was found mediated through type I interferon-independent interferon-stimulated gene (ISG) expression. However, systems biology behind this immune response is unknown. In this study, in vivo transcriptome profiling of PBMC subsets (CD4+, CD8+, CD14+, CD21+, and CD335+) in vaccinated goats and sheep (at 5 days postvaccination) was done to understand this systems biology. Though there are a few differences in the systems biology across cells (specially the NK cells) between sheep and goats, the coordinated response that is inclusive of all the cell subsets was found to be toward the induction of a strong innate immune response, which is needed for an appropriate adaptive immune response.

Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples.

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

Single-cell peripheral immunoprofiling of Alzheimer's and Parkinson's diseases.

Peripheral blood mononuclear cells (PBMCs) may provide insight into the pathogenesis of Alzheimer's disease (AD) or Parkinson's disease (PD). We investigated PBMC samples from 132 well-characterized research participants using seven canonical immune stimulants, mass cytometric identification of 35 PBMC subsets, and single-cell quantification of 15 intracellular signaling markers, followed by machine learning model development to increase predictive power. From these, three main intracellular signaling pathways were identified specifically in PBMC subsets from people with AD versus controls: reduced activation of PLCγ2 across many cell types and stimulations and selectively variable activation of STAT1 and STAT5, depending on stimulant and cell type. Our findings functionally buttress the now multiply-validated observation that a rare coding variant in PLCG2 is associated with a decreased risk of AD. Together, these data suggest enhanced PLCγ2 activity as a potential new therapeutic target for AD with a readily accessible pharmacodynamic biomarker.

T Cell Responses to Neural Autoantigens Are Similar in Alzheimer's Disease Patients and Age-Matched Healthy Controls.

Alzheimer’s disease (AD), a chronic multifactorial and complex neurodegenerative disorder is a leading cause of dementia. Recently, neuroinflammation has been hypothesized as a contributing factor to AD pathogenesis. The role of adaptive immune responses against neuronal antigens, which can either confer protection or induce damage in AD, has not been fully characterized. Here, we measured T cell responses to several potential antigens of neural origin including amyloid precursor protein (APP), amyloid beta (Aβ), tau, α-synuclein, and transactive response DNA binding protein (TDP-43) in patients with AD and age-matched healthy controls (HC). Antigen-specific T cell reactivity was detected for all tested antigens, and response to tau-derived epitopes was particularly strong, but no significant differences between individuals with AD and age-matched HC were identified. We also did not observe any correlation between the antigen-specific T cell responses and clinical variables including age, gender, years since diagnosis and cognitive score. Additionally, further characterization did not reveal any differences in the relative frequency of major Peripheral Blood Mononuclear Cells (PBMC) subsets, or in the expression of genes between AD patients and HC. These observations have not identified a key role of neuronal antigen-specific T cell responses in AD.

A high number of PD-L1+ CD14+ monocytes in peripheral blood is correlated with shorter survival in patients receiving immune checkpoint inhibitors.

Targeting of anti-programmed cell death protein-1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) is a standard therapeutic strategy for various cancers. The aim of the present study was to investigate the prognostic effect of pretreatment PD-L1 expression levels in peripheral blood mononuclear cell (PBMC) subsets for patients with several cancer types receiving anti-PD-1 blockade therapies. Thirty-two patients undergoing anti-PD-L1 blockade therapy, including 15 with non-small cell lung cancer, 14 with gastric cancer, 1 with melanoma, 1 with parotid cancer, and 1 with bladder cancer, were recruited for the present study. PD-L1 expression levels in CD3+, CD4+, CD8+, CD45RA+ and CCR7+ T cells; CD20+ B cells; CD14+ and CD16+ monocytes were measured via flow cytometry before treatment. The percentages of PD-L1+ cells in respective PBMC subsets were compared with respect to different clinicopathological conditions and the association with overall survival (OS) was assessed. The percentages of PD-L1+ with CD3+, CD4+ and CD8+ T cells including naïve and memory T cell subsets, or CD20+ B cells during pretreatment were not markedly correlated with the OS of patients (p > 0.05); however, the percentage of the PD-L1+ CD14+ monocyte subset was significantly correlated with OS (p = 0.0426). Increase in pretreatment expression levels of PD-L1 on CD14+ monocytes is associated with the OS of patients treated with immune checkpoint inhibitors. Further evaluation of large sample size and each specific cancer type might clarify the predictive role of PBMC in patients.

Immunophenotyping Reveals No Significant Perturbation to PBMC Subsets When Co-cultured With Colorectal Adenocarcinoma Caco-2 Cells Exposed to X-Rays.

In vitro co-culture models between tumor cells and peripheral blood mononuclear cells (PBMCs) allow studying the interplay between these cell populations, potentially gaining insight into the in vivo response of the immune system to the presence of the tumor, as well as to possible other agents as radiation used for therapeutic purposes. However, great care is needed in the experimental optimization of models and choice of conditions, as some setups might offer a limited possibility to capture subtle immune perturbations. A co-culture model of PBMCs from healthy donors and colorectal adenocarcinoma Caco-2 cells was successfully adopted in a previous work to measure effects on Caco-2 and modulation of signaling when these latter are irradiated. We here tested if the same experimental setting allows to measure perturbations to the main PBMC subsets: we performed immunophenotyping by means of flow cytometry and quantified helper and cytotoxic T cells, NK cells, and B cells, when PBMCs are cultured alone (control), in presence of non-irradiated Caco-2 cells or when these latter are exposed to a 10 Gy X-ray dose from a conventional radiotherapy accelerator. To measure a baseline response in all experimental conditions, PBMCs were not further stimulated, but only followed in their time-evolution up to 72 h post-irradiation of Caco-2 and assembly of the co-culture. In this time interval PBMCs maintain a high viability (measured via the MTT assay). Caco-2 viability (MTT) is slightly affected by the presence of PBMCs and by the high radiation dose, confirming their radioresistance. Immunophenotyping results indicate a large inter-individual variability for different population subsets already at the control level. We analyzed relative population changes and we detected only a small but significant perturbation to cytotoxic T cells. We conclude that this model, as it is, is not adequate for the measurements of subtler immune perturbations (if any, not washed-out by inter-individual differences). For this purpose, the model needs to be modified and further optimized e.g., including a pre-treatment strategy for PBMCs. We also performed a pooled analysis of all experimental observations with principal component analysis, suggesting the potential of this tool to identify subpopulations of similarly-responding donors.

Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants

BackgroundThis study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids. MethodsA differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously. ResultsThe screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%. ConclusionThis study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.

Syncytin-1/HERV-W envelope is an early activation marker of leukocytes and is upregulated in multiple sclerosis patients.

Syncytin-1 is the envelope protein of the human endogenous retrovirus W (HERV-W). It has been related to multiple sclerosis (MS) but its role in cellular immunity and its pathogenic mechanism in the autoimmune context are not fully understood. We analyzed syncytin-1 levels in peripheral blood mononuclear cells (PBMC) subsets from healthy donors, MS patients in relapse or remission, and patients with acute infections by flow cytometry. PBMC cultures were also prepared to analyze protein expression kinetics. MS patients had higher levels of syncytin-1 levels than controls. We found that syncytin-1 is elevated in monocytes during MS relapses and infections. Cells expressing syncytin-1, including monocytes, T and B lymphocytes, and NKs presented mainly an activated phenotype and, upon stimulation with LPS, its levels increased rapidly on antigen-presenting cells. Syncytin-1 ligation promoted the activation of monocytes, as demonstrated by the upregulation of CD80 and the nonclassical subset CD14low CD16+ . Our results suggest an important role for syncytin-1 in the activation of leukocytes. Given that the expression of syncytin-1 is upregulated in MS patients, this protein might be contributing to the autoimmune cascade in the disease.

Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7+ and IL-6+ T cells was detected in early MS-PBMCs, whereas NFAT1hiT-bethiCD4+ T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7+ and MIPβhi HLA-DR+ lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141hiIRF8hiCXCR3+CD68− dendritic cells. Unlike in Crohn’s disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS.

Platelet and Immune Characteristics of Patients with Immune Thrombocytopaenia Non Responders to Therapeutic Treatments

Introduction: Mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) appear to be multifactorial: autoantibodies, autoreactive CD8+ cytotoxic T cells, enhanced apoptosis and loss of sialic acid which mediates platelet clearance through the Ashwell-Morell receptors present in hepatocytes. Differential involvement of each of them might condition the ability of patients with ITP to respond to treatments. We aimed to examine platelet features and the immunological state of patients with ITP who do not respond to any treatment to detect the unique characteristics of this group. Methods: This was an observational, prospective and transversal study. Patients with chronic primary ITP were included: 28 ITP patients without treatment for at least 6 months (UT-ITP); 36 responders to agonists of thrombopoietin receptors (TPO-RA); and 14 ITP patients who did not respond to first- and second-line treatments (NR-ITP). A healthy control group (n=104) was also included in the study. Active caspase-3, -7, -8 or -9 were determined by flow cytometry using CaspaTag kits (Millipore, Madrid, Spain) in PRP diluted with HEPES-buffer containing 2 mM Ca2+ and 2 mM Gly-Pro-Arg-Pro (Sigma-Aldrich, Madrid, Spain) to prevent fibrin formation . Platelet surface glycan exposure was analysed by determining the binding of lectins by flow cytometry. To do so, washed platelets were incubated with 1 μg/ml Alexa fluor 488-conjugated wheat germ agglutinin lectin (WGA, Invitrogen, Spain) or with 1 μg/ml FITC-conjugated Ricinus communis agglutinin (RCA, Vector Labs, UK). WGA binds to sialic acid and N-acetylglucosaminyl residues, and RCA is a galactose-specific legume lectin which binding serves as an indirect measurement of the loss of sialic acid. Peripheral blood mononuclear cells (PBMCs) subsets were analysed by flow cytometry using specific antibodies. Experimental data was analysed using SPSS 9.0 software (SPSS Inc., Chicago, IL). Results: Platelets from TPO-RA treated and from NR-ITP patients had increased caspase-3, -7, -8 and -9 activities (Figure 1A). Platelets from NR-ITP patients exposed less sialic acid and more N-acetylglucosaminyl residues than the other groups (Figure 1B). Binding of WGA and RCA correlated with caspase activities (Table 1). Distribution of lymphocytes, monocytes and natural killer cells is shown in Table 1. NR-ITP patients had an increased proportion of B lymphocyte (LB), maybe due to a significant rise in the fraction of naive LB cells, and a diminution in LTreg subset. Whereas classical monocytes was increased, nonclassical monocyte fraction was decreased in the UT-ITP and NR-ITP groups. NR-ITP patients also presented an increased CD16+CD56bright cells fraction and a diminished NK CD16+CD56dim subset. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls (monocyte and NK cells subset distribution and LTreg count similar to control group). It is of interest to note the relationship between loss of sialic acid from platelet surface glycans and Tregs count: the most reduced surface exposure of sialic acid, the less Treg count (Figure 2). Conclusions: Platelets from NR-ITP patients had more signs of apoptosis and a different composition of surface glycans, accompanied by a diminished LTreg population, a higher LB naïve percentage, and an increased CD16+CD56bright cells fraction in circulation, indicating a severe deregulation of the immune system. Since an inverse correlation was observed between loss of sialic acid and LTreg count, a potential relationship between glycan composition on the platelet surface and immune response is suggested, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP. On the other hand, TPO-RA appears to have a beneficial effect on immune response. Nevertheless, one of the limitations of our study was that patients were recruited once the response to TPO-RA was achieved; therefore, a longitudinal study would provide more information regarding TPO-RA effects. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Álvarez Roman: Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau. Fernandez-Bello:Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Martín:SOBI: Research Funding; Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. Canales:Novartis: Honoraria; Takeda: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Celgene: Honoraria; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Janssen: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

Impact of fingolimod on CD4+ T cell subset and cytokine profile of relapsing remitting multiple sclerosis patients

Fingolimod inhibits the egress of lymphocytes from lymphatic tissues and also directly affects their functions by modulation of the sphingosine-1-phosphate receptor 1 (S1P1). Our aim was to evaluate the impact of fingolimod on diverse CD4+ T cell subsets, and cytokines.Sixty-six relapsing remitting multiple sclerosis (RRMS) patients were treated with oral fingolimod (0.5 mg) for 6 months, and blood samples were collected at baseline, 3 months, and 6 months. Serum levels of seven cytokines and five chemokines were measured by multiplex immunoassay, and frequencies of peripheral blood mononuclear cell subsets were assessed by flow cytometry, and compared with those of 60 healthy controls.CCL2 (p = 0.039), and CCL5 (p = 0.001) levels were significantly higher in fingolimod-treated patients than healthy controls, whereas end-of-study serum levels of IL-6, IL-8, IL-17A, IL-22, IL-23, TNF-α, CXCL10, and CXCL13 were comparable to the baseline levels. Six months of fingolimod treatment reduced CD3+ T cell (mean ± standard deviation, 72.9% ± 5.5 vs. 60.1% ± 11.1, p < 0.001), CD4+ T cell (62.2% ± 8.5 vs. 24.6% ± 12.9, p < 0.001), CD4+CD25hi regulatory T cell (Treg) (3.4% ± 1.3 vs. 2.0% ± 1.4, p < 0.01), and CD19+ B cell (13.2% ± 5.8 vs. 5.3% ± 2.7, p < 0.001) frequencies, while CD8+ T cells (31.8% ± 7.8 vs. 57.8% ± 13.2, p < 0.001) were increased, and NK and NKT cells remained unchanged. The proportions of intracytoplasmic IL-4, IL-10, IFN-γ, and TNF-α-producing T cells were increased, whereas IL-17-producing cells remained relatively constant as measured by flow cytometry.Fingolimod appears to primarily diminish lymphocyte subsets involved in antigen presentation (CD19+ B and CD4+ T cells) rather than immune cells (CD8+ T, NK, and NKT cells) in charge of host defense against pathogens. In contrast, a relative increase is observed in pro- and anti-inflammatory cytokine-producing T helper subsets (IFN-γ, TNF-α, IL-4, and IL-10-producing CD4+ T cells), suggesting that effector T cells are suppressed to a lesser degree by S1P1 modulation.

Inhibiting the immunoproteasome's β5i catalytic activity affects human peripheral blood-derived immune cell viability.

Small molecule inhibitors selectively targeting the immunoproteasome subunit β5i are currently being developed for the treatment of autoimmune disorders. However, patients carrying loss‐of‐function mutations in the gene encoding β5i (Psmb8) suffer from the proteasome‐associated autoinflammatory syndromes (PRAAS) emphasizing the need to study pharmacological inhibition of immunoproteasome function in human cells. Here, we characterized the immunomodulatory potential of the selective β5i inhibitor ONX 0914 and Bortezomib, a pan‐proteasome inhibitor, in human peripheral blood mononuclear cells (PBMCs). Both compounds efficiently blocked pro‐inflammatory cytokine secretion in human whole blood and PBMC cultures stimulated with toll‐like receptor (TLR) agonists. Furthermore, the compounds inhibited T cell cytokine production induced by recall antigen CMVpp65 or by polyclonal stimulation. The viability of PBMCs, however, was rapidly decreased in the presence of ONX 0914 and Bortezomib demonstrated by decreased residual cytosolic ATP and increased Annexin V surface binding. Interestingly, HLA‐DR + monocytes were rapidly depleted from the cultures in the presence of ONX 0914 as a β5i‐selective inhibitor and Bortezomib. In conclusion, the anti‐inflammatory potential of β5i‐selective inhibitors is correlating with a cytotoxicity increase in human PBMC subsets ex vivo. Our results provide important insights into the anti‐inflammatory mechanism of action of β5i‐inhibitors which currently hold the promise as a novel therapy for autoinflammatory diseases.

Human Peripheral Blood Mononuclear Cells Express High Levels of the Vitamin a Transport Protein, Stimulated by Retinoic Acid 6 (P19-004-19)

ObjectivesStimulated by retinoic acid 6 (STRA6) was the first to be identified in a novel category of proteins, cytokine signaling transporters, due to its ability to function as both a cell surface receptor and a membrane protein that binds to retinol binding protein facilitating cellular uptake of retinol. Retinol is metabolized to all-trans retinoic acid which functions as a steroid hormone transcription factor ligand to regulate genes generally involved in cellular differentiation. The role of vitamin A, as all-trans retinoic acid, in immunity is well characterized, but the role of STRA6 in mediating cellular uptake of vitamin A in immune cells is not understood. The objective of this research was to characterize the expression of STRA6 on human peripheral blood mononuclear cell (PBMC) subsets. We hypothesized STRA6 would be expressed by all PBMC subsets, but at different intensities. MethodsA convenience sample of volunteers were recruited and PBMC were isolated by density gradient centrifugation. Multicolor flow cytometry was used to identify PBMC subsets and expression of STRA6. Confocal microscopy was used to determine cellular location and lipid raft association of STRA6 on cell lines and isolated PBMC. ResultsAll T cell, natural killer cell, monocyte, and dendritic cell subsets analyzed expressed STRA6. Expression of STRA6 (median fluorescence intensity) was higher on CD303+ and CD141+ dendritic cells compared to CD1c+ dendritic cells, CD8+ T cells compared to CD4+ T cells, CD14+/CD16+ and CD14+/CD16- monocytes compared to CD14-/CD16+ monocytes, and CD56-/CD16+ natural killer cells compared to both CD56+/CD16 variable and CD56-/CD16 dim natural killer cells. Expression was not significantly correlated with age, sex, or body composition. Confocal microscopy showed STRA6 was not associated with GM1 cholera toxin subunit B staining and therefore not associated with lipid raft structures. ConclusionsThese results provide preliminary data in which to target vitamin A signaling, through cellular uptake of retinol, in immune cells to improve immune homeostasis in diverse populations. Funding SourcesFunding for this project was provided by internal university support.

Progressive increase of FcεRI expression across several PBMC subsets is associated with atopy and atopic asthma within school-aged children.

Antigen-specific IgE binds the Fcε receptor I (FcεRI) expressed on several types of immune cells, including dendritic cells (DCs). Activation of FcεRI on DCs in atopics has been shown to modulate immune responses that potentially contribute to asthma development. However, the extent to which DC subsets differ in FcεRI expression between atopic children with or without asthma is currently not clear. This study aimed to analyse the expression of FcεRI on peripheral blood mononuclear cells (PBMCs) from atopic children with and without asthma, and non-atopic/non-asthmatic age-matched healthy controls. We performed multiparameter flow cytometry on PBMC from 391 children across three community cohorts and one clinical cohort based in Western Australia. We confirmed expression of FcεRI on basophils, monocytes, plasmacytoid and conventional DCs, with higher proportions of all cell populations expressing FcεRI in atopic compared to non-atopic children. Further, we observed that levels of FcεRI expression were elevated across plasmacytoid and conventional DC as well as basophils in atopic asthmatic compared to atopic non-asthmatic children also after adjusting for serum IgE levels. Our data suggest that the expression pattern of FcεRI on DC and basophils differentiates asthmatic from non-asthmatic atopic children. Given the significant immune modulatory effects observed as a consequence of FcεRI expression, this altered expression pattern is likely to contribute to asthma pathology in children.

Activation of Toll-like Receptor 2 (TLR2) induces Interleukin-6 trans-signaling

Signaling of the pleiotropic cytokine Interleukin-6 (IL-6) via its soluble IL-6R (sIL-6R) has been termed trans-signaling and is thought to be responsible for the pro-inflammatory properties of IL-6. The sIL-6R can be generated by alternative mRNA splicing or proteolytic cleavage of the membrane-bound IL-6R. However, which stimuli induce sIL-6R release and which endogenous signaling pathways are required for this process is poorly understood. Here, we show that activation of Toll-like receptor 2 (TLR2) on primary human peripheral blood mononuclear cells (PBMCs) and on the monocytic cell line THP-1 induces expression and secretion of IL-6 and the generation of sIL-6R. We show by flow cytometry that monocytes are a PBMC subset that expresses TLR2 in conjunction with the IL-6R and are the major cellular source for both IL-6 and sIL-6R. Mechanistically, we find that the metalloproteases ADAM10 and ADAM17 are responsible for cleavage of the IL-6R and therefore sIL-6R generation. Finally, we identify the Extracellular-signal Regulated Kinase (ERK) cascade as a critical pathway that differentially regulates both IL-6 and sIL-6R generation in monocytes.

Characterization of circulating T-, NK-, and NKT cell subsets in patients with colorectal cancer: the peripheral blood immune cell profile

ObjectiveAs the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data.MethodsUsing multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56−), CD56dim NK cells (CD3−CD56dim), CD56bright NK cells (CD3−CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors.ResultsCRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients.ConclusionThe altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.