Profiling the peripheral immune system in patients with autoimmune diseases by mass cytometry (CyTOF)

In contrast to type 1 diabetes (T1D), immune deviations in patients with organ-specific autoimmune diseases, e.g. Hashimoto’s thyroiditis (HT), Grave’s disease (GD) and Addison’s disease (AD), have not been extensively characterised. Additionally, the phenotype of peripheral blood mononuclear cells (PBMC) in these diseases has not been simultaneously compared. The recent introduction of Cytometry by Time-Of-Flight (CyTOF) enables simultaneous measurement of up to 40 parameters at single-cell level, leading to a unique resolution to evaluate the immune system. Thus, we utilised CyTOF to describe and compare the phenotype of PBMC from patients with different autoimmune diseases.

Patients with T1D, HT, GD and AD, without concurrent co-autoimmunity, and healthy donors were recruited. Isolated PBMC were cultured unstimulated or with PMA and ionomycin prior to labelling with 32 metal-tagged antibodies. Our in-house CyTOF panel was designed to simultaneously identify major cell lineages as well as other markers to characterise differentiation, activation and functionality of these cell types. After data acquisition, computational nonlinear dimensionality reduction approaches, combined with clustering algorithms, were applied to reduce the complexity of the analysis.

Data from all samples were pooled and t-distributed stochastic neighbor embedding (t-SNE) analysis was applied to generate a 2D-map, where single cells were grouped in 8 major immune populations according to the expression of lineage markers. Unsupervised hierarchical clustering was then applied to each lineage identifying more than 100 distinct subsets with unique marker expression patterns. The PBMC composition from T1D, HT, GD and AD showed distinct disease-associated profiles. Conclusively, mass cytometry is a powerful tool to simultaneously evaluate immune heterogeneity linked to organ-specific autoimmune diseases.