Dariusz Stepniak

Arena Pharmaceuticals (United States)

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Co-Authors

Frits Koning

Leiden University Medical Center

Peter A. van Veelen

Leiden University Medical Center

Jan W. Drijfhout

Leiden University Medical Center

Hilde Cheroutre

La Jolla Institute for Immunology

Castle Funatake

Thermo Fisher Scientific (Israel)

George K. Papadopoulos

Technological Educational Institute of Epirus

Cherlyn Miranda

AndroScience (United States)

Shourong Li

Anhui Jianzhu University

Nicolas Schrantz

R&D Systems (United States)

Y Kooy

Leiden University Medical Center

Cooperative Institutions

Leiden University Medical Center

Leiden University

AndroScience (United States)

La Jolla Institute for Immunology

University of California, San Diego

Arena Pharmaceuticals (United States)

University of Groningen

Beacon Clinical Research

University of Birmingham

University Medical Center Groningen

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Constitutive intestinal NF-κB does not trigger destructive inflammation unless accompanied by MAPK activation

The Journal of Experimental Medicine (2012)

Mónica Gumá Dariusz Stepniak Helena Shaked Martina E. Spehlmann Steve Shenouda

10.1084/jem.2011024220910c

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Citations (2)

Dominance of an alternative CLIP sequence in the celiac disease associated HLA-DQ2 molecule

Immunogenetics (2008)

Martina Wiesner Dariusz Stepniak Arnoud H. de Ru Antonis K. Moustakis Jan W. Drijfhout

During assembly, HLA class II molecules associate with the invariant chain. As the result, the peptide-binding groove is occupied by an invariant chain peptide termed CLIP (class-II-associated invariant chain peptide; sequence MRMATPLLM). By mass spectrometry, we have now characterized peptides that are naturally present in HLA-DQ2. This analysis revealed that 22 variants of Ii-derived peptides are associated with HLA-DQ2. Strikingly, the large majority of those do not contain the conventional CLIP sequence MRMATPLLM, but instead a peptide that partially overlaps with CLIP, sequence TPLLMQALPM. Peptide binding studies indicate that this alternative CLIP peptide has superior HLA-DQ2 binding properties compared to the conventional CLIP and that the minimal nine-amino-acid binding core consists of the sequence PLLMQALPM, findings that could be corroborated by molecular simulation. The alternative CLIP peptide was also found to be present in HLA-DQ2 molecules isolated from human thymus. Moreover, the alternative CLIP peptide was also found in association with HLA-DQ8. Together, these results indicate that HLA-DQ2 and HLA-DQ8 associate with an alternative CLIP sequence, a property that may relate to the strong association between HLA-DQ molecules and human autoimmune diseases.

Sequence (biology)

10.1007/s00251-008-0310-6

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Citations (18)

Lactoferrin regulates proliferative response of human peripheral blood mononuclear cells to phytohemagglutinin and mixed lymphocyte reaction.

PubMed (2001)

Michał Zimecki Dariusz Stepniak A Szynol Kruzel Ml

Lactoferrin

Mixed lymphocyte reaction

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Citations (17)

CD8aa TCRab intraepithelial lymphocytes MHC restriction (P1462)

The Journal of Immunology (2013)

Florence Lambolez Sofia Mayans Dariusz Stepniak Sakina F. Palida Alexandre Larangé

Abstract T lymphocytes develop in the thymus into two major lineages characterized by expression of ab and gd TCR. Each lineage can be further subdivided into distinct subsets that differ in TCR specificity, phenotype and function. CD8aa TCRab intraepithelial lymphocytes (T-IEL) located in the epithelium of the small intestine is phenotypically different from conventional T cells. CD8aa T-IEL have been studied for years however the question of their MHC restriction has not been elucidated. Therefore, we decided to clone TCR isolated from naturally arising CD8aa T-IEL and retrovirally express these TCRs in BM chimera. First, we cloned four TCRab that were expressed retrovirally in RagKO BM chimera. Analysis of the chimera showed that all TCR clones gave rise to T cells. The T cell that developed were CD4-CD8b- but CD8a+ and phenotypically identical to CD8aa T-IEL isolated from wild type mice. In addition, they were preferentially found in the gut. In order to define the MHC restriction of these particular TCRs, BM chimeras in various MHC-deficient backgrounds were generated. Subsequent analysis of the chimera for the presence of CD8aaTCRab IEL demonstrated that they are dependent either on Kb or Db MHC I or b2m-dependent MHC I aside from KbDb. In conclusion, our results indicate that CD8aa T-IEL develops from precursors expressing particular TCRs that allow their engagement in this unique T cell lineage. In addition, CD8aa T-IEL can harbor clones selected on different MHC molecules.

Chimera (genetics)

Intraepithelial lymphocyte

MHC restriction

10.4049/jimmunol.190.supp.60.14

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Development of two novel microglia-specific monoclonal antibodies to discriminate microglia from other CNS-resident macrophages by flow cytometry

The Journal of Immunology (2020)

Sonja Zahner N. T. Wilkinson Chau Nguyen Jessica Lynn Ellsworth Dariusz Stepniak

Abstract Although microglia are the predominant resident macrophages of the central nervous system (CNS), other macrophages can be found in the CNS. As both populations express CD11b, microglia are generally distinguished from peripheral macrophages by lower expression of CD45. This can introduce bias in the analysis of flow cytometric data and make cell sorting challenging. Other microglia associated markers such as Aif1/Iba1 are less cell specific and therefore of limited use to dissect microglia from other brain residing macrophages. Recent single cell transcriptome studies have identified more specific signature genes of microglia, including surface molecule Tmem119 and transcription factor Sall1. To allow robust discrimination between microglia and other brain resident/infiltrating macrophages based on the expression of a single marker by flow cytometry, we have developed monoclonal rat antibodies against mouse Tmem119 and Sall1, respectively. Both clones, V3RT1GOsz (Tmem119) and NRNSTNX (Sall1), were validated by flow cytometric analysis of fresh mouse brain. Here we show that both antibodies can be used to distinguish microglia from other macrophages by flow cytometry without reliance on CD11b or CD45 expression. The availability of these specific antibodies against microglia signature molecules will contribute significantly to the ability to isolate mouse microglia and their advanced characterization.

Cell Sorting

Forebrain

10.4049/jimmunol.204.supp.86.53

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A sensitive flow cytometric method for multi-parametric analysis of microRNA, messenger RNA and protein in single cells

Methods (2017)

Chunfai Lai Dariusz Stepniak Leslie Sias Castle Funatake

Cell Sorting

Single-Cell Analysis

10.1016/j.ymeth.2017.12.016

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Citations (25)

Novel anti-Mer antibodies in macrophage phenotype analysis (INC2P.419)

The Journal of Immunology (2015)

Dariusz Stepniak Brandi Atteberry Shourong Li Oscar Baterina Natalie Oxford

Abstract Mer is a member of the TAM family of tyrosine kinase receptors that also includes Axl and Tyro3. Mer is expressed on a subset of anti-inflammatory macrophages and is involved in the removal of apoptotic cells. The engulfment of apoptotic cells relies on two soluble accessory molecules Protein S and Gas6 that bind to phosphatidylserine, which is found on the outer leaflet of the plasma membrane of cells undergoing apoptosis. Upon binding these ligands, Mer undergoes autophosphorylation at multiple tyrosine residues that activate the PI3K and Akt pathways. This then leads to the phagocytosis and clearance of apoptotic cells and also results in the direct inhibition of TLR-induced production of pro-inflammatory cytokines. Deficiency of Mer causes general autoimmunity, inflammation, and accumulation of apoptotic bodies. Because Mer expression strongly correlates with macrophage function, It can be used for phenotyping macrophage subpopulations. Here we use novel anti-Mer antibodies in flow cytometric analysis of macrophages and corroborate the expression of this receptor and its importance in distinguishing functional macrophage subsets.

GAS6

MERTK

10.4049/jimmunol.194.supp.55.13

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Subcellular sites for bacterial protein export

Molecular Microbiology (2004)

Nathalie Campo Harold Tjalsma Girbe Buist Dariusz Stepniak Michel Meijer

Summary Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA‐YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the secretory protein pre‐AmyQ, were analysed using green fluorescent protein fusions, immunostaining and/or immunogold labelling techniques. It is shown that SecA, SecY and (pre‐)AmyQ are located at specific sites near and/or in the cytoplasmic membrane of Bacillus subtilis. The localization patterns of these proteins suggest that the Sec machinery is organized in spiral‐like structures along the cell, with most of the translocases organized in specific clusters along these structures. However, this localization appears to be independent of the helicoidal structures formed by the actin‐like cytoskeletal proteins, MreB or Mbl. Interestingly, the specific localization of SecA is dynamic, and depends on active translation. Moreover, reducing the phosphatidylglycerol phospholipids content in the bacterial membrane results in delocalization of SecA, suggesting the involvement of membrane phospholipids in the localization process. These data show for the first time that, in contrast to the recently reported uni‐ExPortal site in the coccoïd Streptococcus pyogenes , multiple sites dedicated to protein export are present in the cytoplasmic membrane of rod‐shaped B. subtilis .

MreB

Immunogold labelling

Transport protein

10.1111/j.1365-2958.2004.04278.x

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Citations (149)

The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses

Proceedings of the National Academy of Sciences (2003)

Willemijn Vader Dariusz Stepniak Y Kooy Luisa Mearin Allan Thompson

In patients with celiac disease, inflammatory T cell responses to HLA-DQ2-bound gluten peptides are thought to cause disease. Two types of HLA-DQ2 molecules exist, termed HLA-DQ2.5 and HLA-DQ2.2. Whereas HLA-DQ2.5 predisposes to celiac disease, HLA-DQ2.2 does not. We now provide evidence that the disease-associated HLA-DQ2.5 molecule presents a large repertoire of gluten peptides, whereas the non-disease-associated HLA-DQ2.2 molecule can present only a subset of these. Moreover, gluten presentation by HLA-DQ2 homozygous antigen-presenting cells was superior to presentation by HLA-DQ2/non-DQ2 heterozygous antigen-presenting cells in terms of T cell proliferation and cytokine secretion. Gluten presentation by HLA-DQ2.5/2.2 heterozygous antigen-presenting cells induced intermediate T cell stimulation. These results correlated with peptide binding to the antigen-presenting cells. Finally, we demonstrate that HLA-DQ trans dimers formed in HLA-DQ2.5/2.2 heterozygous individuals have properties identical with HLA-DQ2.5 dimers. Our findings explain the strongly increased risk of disease development for HLA-DQ2.5 homozygous and HLA-DQ2.2/2.5 heterozygous individuals, and they are indicative of a quantitative model for disease development, where HLA-DQ expression and the available number of T cell-stimulatory gluten peptides are critical limiting factors. This model may have important implications for disease prevention.

HLA-DQ

10.1073/pnas.2135229100

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Citations (372)

Identificatie van de moleculaire pathways in coeliakie middels een functionele genomics aanpak

Frits Koning T.N. Wijmenga A.J. Monsuur Dariusz Stepniak

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