054 Highly Multiplexed Immunophenotyping of Dermatomyositis Skin Lesions

Abstract

Dermatomyositis(DM) is an autoimmune systemic disease that most often affects skin and muscle. The pathogenesis of cellular skin inflammation has yet to be investigated. Previous work revealed a type 1 interferon gene signature characterized predominantly by interferon-beta (β). To investigate the type 1 interferon signature, we identified pathways and cellular phenotypes in a subset of DM patients. 5 Healthy control (HC) and 5 DM formalin-fixed, paraffin-embedded (FFPE) samples obtained from trunk, arm, or leg were stained with a panel of 35 metal conjugated antibodies. Regions of interest (ROI) of 500x800μm were ablated at a frequency of 200Hz on the Hyperion Imaging System (Fluidigm). The resulting files were MCD files were exported to 16-bit TIFF files using MCD ViewerTM (Fluidigm). Cell segmentation was performed using an app-based algorithm in Visiopharm. Per object mean pixel intensity (MPI) was gathered and analyzed using histoCAT. Positive and negative cell populations were identified using a sliding scale for each channel. Phenograph algorithm was used for unsupervised clustering of cell populations after thresholding each channel. Statistical analysis between groups was performed using the Mann-Whitney test all values reported as mean ± SEM. Skin lesions of DM patients contain an increased number of CD163+ cells compared to normal skin from HC patients (14±4.7 vs 2±1 cells/ROI) p<0.05). CD163+ cells had increased MPI of key inflammatory pathways: pSTING (34.4±4.9 vs 3.9±1.9), IFNβ (8.2±0.8 vs 4.4±7.5), and IL17 (6.5±1.0 vs 1.3±0.3); all p<0.05. A population of CD4 cells was identified that produced higher IFNβ MPI compared to HC CD4 cells (16.4±4.5 vs 8.0±0.8; p<0.01). Lesional DM skin also contained more FOXP3+ CD4 cells when compared to HC (64±20.3 vs 6±1.3 cells/ROI; p<0.05). The function of these cells is unclear. Compared to HC CD163+ cells in DM appear to be an important source of IFNb via activation of the STING pathway. IFNβ is produced by both CD163+ and a subset of CD4 cells.