Session Type: Poster Session (Tuesday)
Session Time: 9:00AM-11:00AM
Background/Purpose: The immune infiltrate of skin lesions of Cutaneous Lupus Erythematosus (CLE) patients is known to be rich and complex. Single cell RNAseq (scRNAseq) is a unique approach to understand the cell-specific transcriptional heterogeneity in a complex tissue. ScRNAseq analyses of CLE skin biopsies has the unique potential to improve our understanding of human disease tissues and provide critical biological insights into the CLE pathogenesis.
Methods: Skin punch biopsies were obtained from healthy skin or lesional skin from CLE patients. Skin storage conditions and tissue dissociation protocol were optimized using healthy skin biopsies. RNAseq protocol was tested with blood-sorted pDCs and monocytes. Skin biopsies were collected from patients with DLE or SCLE. After tissue dissociation and cell staining, single CD45+ immune cells were sorted using flow cytometry and scRNAseq was performed using the SMART-seq2 protocol for cDNA synthesis and library preparation prior to sequencing.
Results: The scRNAseq protocol showed a similar sensitivity as a state-of-the-art published protocol (Villani AC et al, Science 2017). Avoiding cryopreservation and digestion of the skin biopsies with collagenase provided the best immune cell yield. Approximatively 85% of the isolated single cells produced RNAseq data that passed quality control. Analysis of 5 biopsies provided transcriptomic characterization of B-cells, CD4 and CD8 positive T-cells, Macrophages, Dendritic cells, and pDCs from skin lesions and suggested immune infiltrate heterogeneity in CLE patients. RNAseq data was also leveraged to define cell type marker panel for scqPCR which allowed a more focused cost- and time- effective approach for follow-up/validation of gene expression analyses.
Conclusion: We developed a protocol that successfully identified and characterized the immune infiltrate from skin punch biopsies of CLE patients. Analyses from additional patients will be needed to further understand the immunological heterogeneity of CLE skin lesions.
To cite this abstract in AMA style:Gardet A, Carlile T, Chou W, Li K, Pellerin A, Challa R, Chen W, Sun C, Franchimont N, Werth V, Rabah D. Comprehensive Characterization of the Immune Infiltrate of Skin Biopsies from Cutaneous Lupus Erythematosus Patients Using Single Cell RNAseq [abstract]. Arthritis Rheumatol. 2019; 71 (suppl 10). https://acrabstracts.org/abstract/comprehensive-characterization-of-the-immune-infiltrate-of-skin-biopsies-from-cutaneous-lupus-erythematosus-patients-using-single-cell-rnaseq/. Accessed December 5, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/comprehensive-characterization-of-the-immune-infiltrate-of-skin-biopsies-from-cutaneous-lupus-erythematosus-patients-using-single-cell-rnaseq/