Background/Purpose: Systemic sclerosis (SSc), is a chronic autoimmune disorder of the connective tissue. There is an unmet need to define precise immune correlates involved in SSc pathogenesis. The interplay between the  tissue microenvironment such as the skin and the periphery in potentiating an inflammatory response remains to be elucidated. This knowledge gap is a major impediment for development of therapeutic interventions.

Methods: Our Immunomics platform integrates highly multiplexed technologies with the objective of identifying unique immune signatures that cluster meaningfully at the single-cell level. 1) Gene expression studies using Nanostring – Human dermal fibroblasts stimulated with culture supernatants of blood and skin derived DNA Topoisomerase I (Topo I, an autoantigen relevant in SSc ) specific T cell lines (TCLs)  2) Mass spectrometry (CyTOF) analysis of peripheral blood using 35+ phenotypic and intracellular markers. 3) Next-gen, RNA-seq analysis of the the whole transcriptome, including TCR  usage of Topo I specific TCLs. Using this approach, we interrogated samples from SSc subjects stratified (according to modified Rodnan skin score, MRSS) as patients with ILD (n=13), No ILD (n=13) and compared to healthy controls (n=8). The dense data sets generated from these high throughput measurements were analysed using computational tools such as Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE), Principal Component Analysis (PCA) and Ingenuity Pathway Analysis (IPA).

Results: Skin derived Topo I specific TCLs expressed significantly higher amounts of IL-17A and IL-13 (p value < .001) compared to blood derived Topo I specific TCLs. Nanostring gene expression studies revealed that IL-1a, IL-1β, IL-6, IL-8, IL-11, CCL2, CXCL2, CXCL10, CSF3 and TNFSF14 were significantly (p value < .05) in the human dermal fibroblasts stimulated with culture supernatants from skin-derived TCLs. Gene expression was  also confirmed at the protein level using Enzyme linked immunosorbent assay (ELISA). IPA gene ontology studies annotated IL-17A as a top upstream regulator of these genes. CyTOF analysis showed that CD4+CD45RO+CD161+CCR4+CCR6+ Th17 cells were increased (p value < .03) in SSc subjects with No ILD compared to patients with ILD and healthy controls. Finally, unique clonotypes of Topo I specific CD4+ T cells could be identified. 

Conclusion: We have defined an Immunome in SSc that circumscribes an altered frequency of Th17 cell subset in patients with ILD as compared to No ILD. IL-17A was identified as the top regulator of genes upregulated by Topo I specific T cells in human dermal fibroblasts. Interestingly, cytokines encoded  by some of  these genes such as IL-1a, IL-1β, IL-6 and IL-11 are known to be involved in Th17 polarisation. We hypothesise that skin homing Topo I specific T cells secreting IL-17A upregulates inflammatory cytokines in SSc skin, perpetuating inflammation and polarizing a cutaneous Th17 response. These results annotate an interplay between the skin microenvironment and the periphery in shaping the Immunome relevant in SSc pathogenesis

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-multidimensional-immunomics-approach-annotates-an-immunome-shaped-by-the-interplay-between-the-periphery-and-the-skin-microenvironment-in-systemic-sclerosis/