Creation of Panels with Utility for clinical studies of Patients with Systemic Autoimmune Diseases (SADs)
Paulina Rybakowska1, Concepción Marañón1, and Marta E. Alarcón-Riquelme1,2
1Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO); 2Institure for Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
Background/Goals: Mass cytometry (HELIOS) offers the possibility of studying immune networks at a single cell level with approximately 40 probes targeting different markers. Therefore, different population landscapes and their functional responses can be studied. For this reasons this technology is the best for identifying cells and pathways involved in the pathogenesis of heterogeneous and overlapping diseases like SADs. The PRECISESADS project aims at the molecular re-classification of the SADS combining molecular and cellular "-omics" considering all the diseases jointly and not as separate entities.
The first part of this investigation was to optimize the methodology in order to study clinical samples of SADs using the HELIOS instrument.
Methods: A panel of surface markers and intracellular cytokines were designed using MaxPar panel designer. Clone validation of antibodies was performed by conventional flow cytometry and in-house conjugation to the metal isotopes was carried out using Maxpar antibody labeling kit. Whole blood was stimulated with various stimuli: LPS (TLR4), poly I:C (TLR3), R848 (TLR7/8) and R837 (TLR7). Part of the sample was frozen, using different stabilizing methods and stored at -80ºC until acquisition. The other part was stained for surface and cytokine markers and acquired on HELIOS. Conventional biaxal plot gating and the SPADE algorithm are used for data analysis.
Results: A panel for surface and intracellular cytokines was specially designed for the PRECISESADS study. Antibody clones were validated and conjugated to the metal isotopes. Surface and cytokine panels were combined and cytokine production was detected across different stimuli and different cell populations in HELIOS.
Summary: We build the basis for future studies using mass cytometry for cellular and molecular re-classification of SADs.
Funding: PRECISESADS project Grant No. 115565
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