4 research outputs found

    Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood

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    Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings

    Identificatie van MHC klasse I geassocieerde antigeenpeptiden. Ontwikkeling van gevoelige massaspectrometrische sequentie analysetechnieken

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    Betreft de ontwikkeling van gevoelige microkolom HPLC-ESI/MS methoden en technieken voor de analyse van MHC klasse I geassocieerde antigeenpeptiden. De analyse bestaat uit de identificatie van T-cel stimulerende peptiden in MHC elutiemengsels van geinfecteerde celkweekculturen gevolgd door de bepaling van de aminozuurvolgorde (sequentie) van deze peptiden. Elutiemengsels van MHC moleculen kunnen zeer grote aantallen (tot 2000) verschillende peptiden bevatten met sterk wisselende samenstelling afhankelijk van te onderzoeken cellijnen of weefselmateriaal. Beschikbare hoeveelheden van individuele peptiden in dergelijke mengsels voor onderzoek liggen in de femtomol (10 exp. -10) tot picomol (10 exp. -12) range per miljard cellen. HPLC-ESI/MS wordt als de meest geschikte techniek beschouwd voor deze toepassing. In dit onderzoek is microkolom HPLC schakeltechniek ontwikkeld ; de condities zijn aangepast voor een optimale gevoeligheid in ESI-MS. De hiermee behaalde gevoeligheden liggen tussen 10 en 50 fmol voor identificatiedoeleinden (molecuulgewichtbepaling), afhankelijk van aard en soort peptide. Sequentie-analyses vergen circa tien keer grotere hoeveelheden (100-500 fmol/peptide). Dit komt overeen met de hoeveelheden antigeenpeptiden in middelgrote celkweken (10 exp. 10 cellen).The development of methods and techniques for the sensitive analysis of MHC class I associated peptides has been described. Analysis concerns the identification and sequence determination of specific T-cel stimulating antigenic peptides in complex mixtures derived from cell cultures. Such mixtures comprise a few hundreds to thousand different class I peptides present at femtomol (10 exp. -15) to picomol (10 exp. -12) level per billion cells. Liquid chromatography-electrospray ionisation mass spectrometry has emerged as the most appropriate technique for such analysis. For this purpose, a micro-column switching technique has been developed; conditions were optimised directed to large volume sample introduction, concentration and separation for their sensitive analysis at suitable ESI conditions. The sensitivity of the method for molecular weight determination (identification) range between 10 and 50 fmol for individual peptides. Sequence analysis required a ten-fold higher amount of peptide, but is sufficiently low for application to amounts that can be made available from medium size cell cultures of typically 10 exp. 10 cells.RIV

    Identification of MHC class I associated peptides. Development of sensitive mass spectrometric sequence analysis techniques

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    Betreft de ontwikkeling van gevoelige microkolom HPLC-ESI/MS methoden en technieken voor de analyse van MHC klasse I geassocieerde antigeenpeptiden. De analyse bestaat uit de identificatie van T-cel stimulerende peptiden in MHC elutiemengsels van geinfecteerde celkweekculturen gevolgd door de bepaling van de aminozuurvolgorde (sequentie) van deze peptiden. Elutiemengsels van MHC moleculen kunnen zeer grote aantallen (tot 2000) verschillende peptiden bevatten met sterk wisselende samenstelling afhankelijk van te onderzoeken cellijnen of weefselmateriaal. Beschikbare hoeveelheden van individuele peptiden in dergelijke mengsels voor onderzoek liggen in de femtomol (10 exp. -10) tot picomol (10 exp. -12) range per miljard cellen. HPLC-ESI/MS wordt als de meest geschikte techniek beschouwd voor deze toepassing. In dit onderzoek is microkolom HPLC schakeltechniek ontwikkeld ; de condities zijn aangepast voor een optimale gevoeligheid in ESI-MS. De hiermee behaalde gevoeligheden liggen tussen 10 en 50 fmol voor identificatiedoeleinden (molecuulgewichtbepaling), afhankelijk van aard en soort peptide. Sequentie-analyses vergen circa tien keer grotere hoeveelheden (100-500 fmol/peptide). Dit komt overeen met de hoeveelheden antigeenpeptiden in middelgrote celkweken (10 exp. 10 cellen).<br

    Sampling from the proteome to the human leukocyte antigen-DR (HLA-DR) ligandome proceeds via high specificity

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    Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease
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