scholarly journals Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. Casasola-LaMacchia ◽  
M. S. Ritorto ◽  
R. J. Seward ◽  
N. Ahyi-Amendah ◽  
A. Ciarla ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dendritic Cells ◽  
Human Leukocyte ◽  
Human Monocyte ◽  
Fold Increase ◽  
Protein Quantification ◽  
Protein Levels ◽  
Hla Dr ◽  
Α Chain ◽  
Hla Dqa1

AbstractThe major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3–7% and ≤ 1%, respectively.

scholarly journals Inhibition of O-GlcNAc Transferase Alters the Differentiation and Maturation Process of Human Monocyte Derived Dendritic Cells

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3312
Author(s):  
Matjaž Weiss ◽  
Marko Anderluh ◽  
Martina Gobec
Keyword(s):  
Dendritic Cells ◽  
Posttranslational Modification ◽  
Human Monocyte ◽  
T Cell Differentiation ◽  
Maturation Process ◽  
Hla Dr ◽  
Glcnac Transferase ◽  
And Function

The O-GlcNAcylation is a posttranslational modification of proteins regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase. These enzymes regulate the development, proliferation and function of cells, including the immune cells. Herein, we focused on the role of O-GlcNAcylation in human monocyte derived dendritic cells (moDCs). Our study suggests that inhibition of OGT modulates AKT and MEK/ERK pathways in moDCs. Changes were also observed in the expression levels of relevant surface markers, where reduced expression of CD80 and DC-SIGN, and increased expression of CD14, CD86 and HLA-DR occurred. We also noticed decreased IL-10 and increased IL-6 production, along with diminished endocytotic capacity of the cells, indicating that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature DCs. Furthermore, the inhibition of OGT altered the maturation process of immature moDCs, since a CD14medDC-SIGNlowHLA-DRmedCD80lowCD86high profile was noticed when OGT inhibitor, OSMI-1, was present. To evaluate DCs ability to influence T cell differentiation and polarization, we co-cultured these cells. Surprisingly, the observed phenotypic changes of mature moDCs generated in the presence of OSMI-1 led to an increased proliferation of allogeneic T cells, while their polarization was not affected. Taken together, we confirm that shifting the O-GlcNAcylation status due to OGT inhibition alters the differentiation and function of moDCs in in vitro conditions.

scholarly journals Hypoxia Shapes Autophagy in LPS-Activated Dendritic Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Sara Monaci ◽  
Carlo Aldinucci ◽  
Daniela Rossi ◽  
Gaia Giuntini ◽  
Irene Filippi ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Human Monocyte ◽  
Bafilomycin A1 ◽  
Adaptive Responses ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Hypoxic Environment ◽  
Tlr4 Ligand ◽  
Autophagic Process ◽  
Lps Treatment

During their lifespan, dendritic cells (DCs) are exposed to different pO2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.

scholarly journals DC-SIGN Is the Major Mycobacterium tuberculosis Receptor on Human Dendritic Cells

2002 ◽  
Vol 197 (1) ◽  
pp. 121-127 ◽  
Author(s):  
Ludovic Tailleux ◽  
Olivier Schwartz ◽  
Jean-Louis Herrmann ◽  
Elisabeth Pivert ◽  
Mary Jackson ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Mycobacterium Tuberculosis ◽  
Human Monocyte ◽  
Mannose Receptor ◽  
Intercellular Adhesion Molecule ◽  
Minor Role ◽  
Hla Dr ◽  
A Minor ◽  
Human Dendritic Cells

Early interactions between lung dendritic cells (LDCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, are thought to be critical for mounting a protective anti-mycobacterial immune response and for determining the outcome of infection. However, these interactions are poorly understood, at least at the molecular level. Here we show that M. tuberculosis enters human monocyte-derived DCs after binding to the recently identified lectin DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). By contrast, complement receptor (CR)3 and mannose receptor (MR), which are the main M. tuberculosis receptors on macrophages (Mϕs), appeared to play a minor role, if any, in mycobacterial binding to DCs. The mycobacteria-specific lipoglycan lipoarabinomannan (LAM) was identified as a key ligand of DC-SIGN. Freshly isolated human LDCs were found to express DC-SIGN, and M. tuberculosis–derived material was detected in CD14−HLA-DR+DC-SIGN+ cells in lymph nodes (LNs) from patients with tuberculosis. Thus, as for human immunodeficiency virus (HIV), which is captured by the same receptor, DC-SIGN–mediated entry of M. tuberculosis in DCs in vivo is likely to influence bacterial persistence and host immunity.

scholarly journals High-Throughput Analysis of Amino Acids for Protein Quantification in Plant and Animal-Derived Samples Using High Resolution Mass Spectrometry

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7578
Author(s):  
Priyanka Reddy ◽  
Aaron Elkins ◽  
Joe Panozzo ◽  
Simone J. Rochfort
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Retention Time ◽  
High Resolution Mass Spectrometry ◽  
Amino Acid Content ◽  
Protein Quantification ◽  
Borate Buffer Solution ◽  
Buffer Solution ◽  
High Throughput Analysis ◽  
Protein Levels

Current methods for measuring the abundance of proteogenic amino acids in plants require derivatisation, extended run times, very sensitive pH adjustments of the protein hydrolysates, and the use of buffers in the chromatographic phases. Here, we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of amino acids that requires only three steps: hydrolysis, neutralisation, and sample dilution with a borate buffer solution for pH and retention time stability. The method shows excellent repeatability (repeated consecutive injections) and reproducibility (repeated hydrolysis) in the amino acid content, peak area, and retention time for all the standard amino acids. The chromatographic run time is 20 min with a reproducibility and repeatability of <1% for the retention time and <11% for the peak area of the BSA and quality control (QC) lentil samples. The reproducibility of the total protein levels in the hydrolysis batches 1–4 was <12% for the BSA and the lentil samples. The level of detection on column was below 0.1 µM for most amino acids (mean 0.017 µM).

scholarly journals IL-1β Induces SOCS2 Expression in Human Dendritic Cells

2019 ◽  
Vol 20 (23) ◽  
pp. 5931
Author(s):  
Muamera Sarajlic ◽  
Theresa Neuper ◽  
Kim Tamara Föhrenbach Quiroz ◽  
Sara Michelini ◽  
Julia Vetter ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Myeloid Leukemia ◽  
Inflammatory Responses ◽  
Human Monocyte ◽  
Cytokine Signaling ◽  
Potent Inducer ◽  
Protein Levels ◽  
Surface Marker Expression ◽  
Human Dendritic Cells

Dendritic cells (DCs) regulate immunity and inflammation and respond to various stimuli, including cytokines. IL-1β is a key cytokine in the course of both acute and chronic inflammatory responses, making it indispensable for protection of the host, but also linking it to several diseases. Thus, IL-1β signaling must be tightly regulated. As suppressor of cytokine signaling (SOCS) proteins effectively control immune responses, we investigated the role of SOCS2 in IL-1β-induced DC activation. Human monocyte-derived DCs were stimulated with IL-1β, and SOCS2 mRNA and protein levels were measured. DC activation was assessed by cytokine secretion and surface marker expression. For functional analysis, small interfering RNA (siRNA)-based SOCS2 silencing was performed. SOCS2 expression was also analyzed in a curated NCBI GEO dataset of myeloid leukemia patients. We found IL-1β to be a potent inducer of SOCS2 expression. By silencing SOCS2, we showed that SOCS2 specifically limits IL-1β-induced IL-8 secretion. Moreover, our analysis revealed that SOCS2 levels are significantly increased in patients with acute and chronic myeloid leukemia, two hematological malignancies where disease progression is closely linked to IL-1β. This study identifies SOCS2 as a novel IL-1β-inducible target gene and points toward a potential role of SOCS2 in IL-1β-mediated DC activation.

scholarly journals EFFECTS OF IN VITRO 1,25 DIHYDROXYVITAMIN D ON MATURATION OF DENDRITIC CELLS IN GRAVES’ DISEASE PATIENTS

2016 ◽  
Vol 9 (5) ◽  
pp. 221
Author(s):  
Dyah Purnamasari ◽  
Samsuridjal Djauzi ◽  
Siti Setiati ◽  
Alida Harahap ◽  
Tjokorda Gde Dalem Pemayun ◽  
...  
Keyword(s):  
Vitamin D ◽  
Dendritic Cells ◽  
Lupus Erythematosus ◽  
Healthy Subjects ◽  
Human Leukocyte ◽  
Graves Disease ◽  
Autoimmune Reaction ◽  
Hla Dr ◽  
Dc Maturation

ABSTRACTObjective: The autoimmune reaction in Graves’ disease (GD) is induced by self-antigen, which is presented by dendritic cells (DCs). DCs in GD have more active immune responses than those in healthy subjects. The ability of DC as antigen-presenting cell is determined by its maturity level. InGD, vitamin D level is inversely proportional to antibody titer and proportionally associated with remission status. Studies on healthy subjects andautoimmune patients (systemic lupus erythematosus (SLE), multiple sclerosis (MS), and Crohn’s disease) have demonstrated immunoregulatoryeffects of vitamin D, mainly through inhibition of DC maturation, which may decrease the DC’s immunogenic profile. This study aims to identify theeffect of 1,25-D3 in vitro on DC maturation in patients with GD.Methods: This is an experimental study, which was conducted in 12 GD patients with thyrotoxicosis. Monocyte-derived DC of GD patients wascultured, with or without 1,25-D3 in vitro at monocytic phase. The DC maturation was then stimulated by lipopolysaccharide (LPS) and evaluatedbased on the expression of DC markers (human leukocyte antigen-D-related [HLA-DR], CD80, CD40, CD83, CD14, and CD206) and the ratio of cytokineinterleukin-12 (IL-12)/IL-10 levels in the supernatants.Results: Following the LPS stimulation, DC with 1,25-D3 showed lower expressions of HLA-DR, CD80, CD40, and CD83, and higher expressions ofCD14 and CD206 compared to DC without 1,25-D3. DC with 1,25-D3 had lower ratio of IL-12/IL-10 levels than those without 1,25-D3.Conclusion: In vitro 1,25-D3 supplementation inhibits DC maturation in patients with GD.Keywords: Vitamin D, Graves’ disease, Dendritic cells.

scholarly journals Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells

2020 ◽  
Author(s):  
Robert Parker ◽  
Thomas Partridge ◽  
Catherine Wormald ◽  
Rebeca Kawahara ◽  
Victoria Stalls ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Antigen Processing ◽  
Human Leukocyte ◽  
Class Ii ◽  
Binding Motif ◽  
S Protein ◽  
Leukocyte Antigen ◽  
Cell Responses ◽  
Hla Dr ◽  
Nested Sets

ABSTRACTUnderstanding and eliciting protective immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent priority. To facilitate these objectives, we have profiled the repertoire of human leukocyte antigen class II (HLA-II)-bound peptides presented by HLA-DR diverse monocyte-derived dendritic cells pulsed with SARS-CoV-2 spike (S) protein. We identify 209 unique HLA-II-bound peptide sequences, many forming nested sets, which map to sites throughout S including glycosylated regions. Comparison of the glycosylation profile of the S protein to that of the HLA-II-bound S peptides revealed substantial trimming of glycan residues on the latter, likely introduced during antigen processing. Our data also highlight the receptor-binding motif in S1 as a HLA-DR-binding peptide-rich region. Results from this study have application in vaccine design, and will aid analysis of CD4+ T cell responses in infected individuals and vaccine recipients.

scholarly journals HLA-DR-presented Peptide Repertoires Derived From Human Monocyte-derived Dendritic Cells Pulsed With Blood Coagulation Factor VIII

2011 ◽  
Vol 10 (6) ◽  
pp. M110.002246 ◽  
Author(s):  
Simon D. van Haren ◽  
Eszter Herczenik ◽  
Anja ten Brinke ◽  
Koen Mertens ◽  
Jan Voorberg ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Factor Viii ◽  
Blood Coagulation ◽  
Coagulation Factor ◽  
Human Monocyte ◽  
Coagulation Factor Viii ◽  
Hla Dr ◽  
Blood Coagulation Factor Viii

scholarly journals Regulatory polymorphisms modulate the expression of HLA class II molecules and promote autoimmunity

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Prithvi Raj ◽  
Ekta Rai ◽  
Ran Song ◽  
Shaheen Khan ◽  
Benjamin E Wakeland ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Fold Increase ◽  
Surface Expression ◽  
Common Disease ◽  
Hla Polymorphism ◽  
Functional Variants ◽  
Hla Dr ◽  
D Region ◽  
Regulatory Complex ◽  
Hla Dqa1

Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus (SLE). Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites. These extensive functional variations are a new and potent facet of HLA polymorphism. Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1, HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner, resulting in a 2.5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes, which increases to over 4-fold after stimulation. Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes.

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