Gábor Szebeni

Immunophenotyping of treatment naive patients with systemic autoimmune diseases by single cell mass cytometry

Gábor J. Szebeni 1, József Ágoston Balog1, Ágnes Zvara1, László Kovács2, Attila Balog2, László G. Puskás1

1Laboratory of Functional Genomics, Biological Research Centre, Hungarian Academy of Sciences; Temesvári krt. 62, Szeged, H-6726, Hungary
2Department of Rheumatology and Immunology, Faculty of Medicine, Albert Szent-Gyorgyi Health Centre, University of Szeged, Szeged, Hungary

INRODUCTION. Epidemiological data highlights the rising incidence of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and systemic sclerosis (SSC) in the developed world over the last decades. Currently available treatments are palliative reducing the symptoms and supporting patient’s wealth. Therefore, studies are much needed to deeply reveal cellular features responsible for pathologies or to identify early diagnostic and prognostic markers associated with RA, SLE and SSC, respectively.

METHODS. We focus on the multiparametric and functional characterization of RA, SLE and SSC using single cell mass cytometry. Treatment-naive patients suffering either in RA, SLE or SSC who had not received non-steroidal anti-inflammatory drugs, disease modifying anti-rheumatic drugs and glucocorticoids until the time of blood sampling are enrolled aligned with age and gender matched healthy individuals. Healthy controls have a negative history of rheumatic symptoms, negative status upon detailed physical and laboratory examination. Human subjects are informed and handled in line with the Declaration of Helsinki and relevant Directives of the EU. PBMCs are purified by Ficoll density gradient centrifugation. CD4+ T-cells are purified by magnetic bead separation for whole transcriptome analysis. PBMCs are labeled by antibodies to detect markers at single cell resolution by mass cytometry. In mass cytometry detection is based on antibodies which are labeled by stable metal isotopes. Thus, autofluorescence and spectral overlapping are eliminated. Markers identified by the transcriptome analysis associated with either RA, SLE or SSC are screened for validation by mass cytometry at protein level.

RESULTS. After gating out the calibration beads and cellular doublets (191Ir+/193Ir+ heterogeneous) live cells (195Pt cisplatin negative) are determined. Using 26 antibodies in one single tube the following sub-populations have been defined within single living CD45+ cells: B-cells, monocytes, CD16+ myeloid dendritic cells, NK-cells, CD4Tcm, CD4Tem, CD4Tnaive, CD4Th1, CD4Th2, CD4Th0, CD8Tcm, CD8Tem, CD8Tnaive, CD8Tc1, CD8Tc2, CD8Tc0. In order to characterize the phenotype of the above mentioned cell types the following markers have been investigated on each subtype: CD5, CD7, CD9, CD28, CD49d, CD161, CXCR3, CD25, CD69, CD27, CD57, CD127. Stochastic neighbor embedding (viSNE) analysis dissects the different immunophenotype of RA, SSC and SLE samples based on the multidimensional cellular relatedness of 26 marker expression at single cell resolution in each disease.

CONCLUSION. Mass cytometry deeply reveals cellular heterogeneity on the basis of highly multiplex phenotypical and functional characterization in RA, SLE and SSC.