Autoimmune diseases often exhibit a puzzling pattern: although harmful immune responses target molecules (autoantigens) found throughout an organ, the resulting inflammation tends to occur in patches rather than affecting the entire organ evenly. For example: In type 1 diabetes, only about 20% of insulin-producing areas (islets) show inflammation, while surrounding tissue looks normal. In cutaneous lupus, the rash occurs in clearly defined areas surrounded by normal skin. We see patchy inflammation in many other autoimmune diseases like psoriasis, ulcerative colitis, giant cell arteritis, and atopic dermatitis. However, the exact mechanisms behind this remain unclear. A key question is what is happening at the boundary between inflamed and unaffected tissue. In response to infections, the immune system carefully balances clearing the threat while preventing excessive inflammation to avoid damaging tissues. We think a similar balance occurs in autoimmune diseases: while some areas show active inflammation, nearby tissues (called perilesional tissue) are partly protected. The mechanisms that protect this perilesional tissue are responsible for the patchy pattern of inflammation we see. In these regions the immune system isn’t entirely normal – it shows a milder form of the immune activity seen in the disease. However, these boundary areas seem to strike a balance that suppresses inflammation and protects the tissue from further harm. In conditions like type 1 diabetes, this protective balance can eventually fail, leading to significant damage to the pancreas and the loss of insulin-producing cells. The same occurs in other autoimmune diseases like Sjogren’s syndrome (causing dry eyes and dry mouth) and Hashimoto’s thyroiditis (causing hypothyroidism). In other autoimmune conditions like systemic lupus erythematosus, rheumatoid arthritis and vasculitis inflammatory varies in intensity over time. Individuals experience periods of remission interspersed by flare. During periods of remission we think that, rather than tissues returning completely to normal, similar protective mechanisms keep inflammation settled. We would like to understand these protective mechanisms. This is essential to prevent organ damage and preserve function or to keep disease in remission. If we understood them better we may be able to boost them in people at risk of autoimmune disease to avoid tissue damage or make sure that the disease remains settled. We propose two different ways to examine this: Single-cell RNA sequencing (scRNAseq): This is a technique that looks at what individual cells are making (RNA). We can use this to tell us what a cell is and what it is doing. This has been used in many autoimmune diseases before although not to answer this specific question. Much of this data is available for researchers to reuse. We will use this to look at perilesional tissue and healed tissue in multiple diseases and look for common patterns. Spatial transcriptomics (ST): This technique uses microscope slides from tissue to measure what a cell is making (RNA). The advantage of this approach is that we also know where the cell is in tissue and can see how close it is to areas of inflammation. We aim to use this to identify the mechanisms that maintain the protective balance in tissues. We will initially apply this approach in type 1 diabetes and lupus rashes. The ultimate goal is to uncover universal strategies that tissues use to prevent recurrent and spreading inflammation. This could lead to innovative treatments that protect tissue from damage or restore the immune balance without long-term dependency on medications.

Autoimmune diseases share many commonalities including genetic predisposition, response to the same treatment and co-occurrence. Autoimmune inflammation is temporally and spatially restricted, marked by cycles of flare and remission, and unevenly distributed throughout the affected organ as a result of homeostatic, tolerizing mechanisms in tissue that promote resolution and prevent organ damage. A shared disease feature is that while autoantigens are ubiquitously expressed within the affected organ, inflammation is often patchy. In recent onset type I diabetes (T1D), only around one-fifth of islets are inflamed and enclosed by CD8+ T cells with surrounding tissue having a normal appearance. In clinically unaffected skin adjacent to lesional cutaneous lupus, there is infiltration of dendritic cells and a broad interferon signature across cell types that distinguishes it from truly normal skin. Across these studies there is evidence of an attenuated immune response in perilesional tissue resembling overt disease. Indeed, postinflammatory or perilesional tissue is not the same as unaffected tissue; it is marked by alterations in cellular composition, tissue architecture, and cytokine profile. It often contains the same immune cell subsets, and transcriptional programs but with reduced abundance and gene expression of certain pathogenic programs. These changes are required to prevent recurrence or prevent organ damage, maintain tissue tolerance, and restrain self-reactive responses. Another shared disease feature is that they often flare in the same anatomical site; skin diseases recur in the same location, arthritis affects the same joints, multiple sclerosis frequently relapses at the same site. In barrier sites such as skin, an initial inflammatory insult, such as an infection, results in the development of resident memory T cells (Trm) to allow rapid recall. These Trm responses are particularly well characterized in skin diseases (e.g., vitiligo, cutaneous lupus) but have also been demonstrated in non-barrier organs, such as in T1D pancreas and in the central nervous system of multiple sclerosis patients. Treatment can inhibit Trm responses but equally disease can resolve and remain inactive without treatment or following its withdrawal, highlighting the activity of endogenous mechanisms that prevent recurrence that have yet to be uncovered. Altogether, there are likely shared mechanisms of tissue tolerance underlying these shared disease patterns of patchy organ involvement and relapsing diseases. We hypothesize that there are active immune mechanisms that inhibit inflammation in both healed and perilesional tissues that are conserved across autoimmune diseases and that insights from studying these tissues can inform our understanding of what constitutes remission in autoimmunity and support the development of tailored tolerogenic therapies in future. These mechanisms have been difficult to characterize because ostensibly healthy tissue is rarely biopsied and analysis of bulk or dissociated tissue lacks the spatial resolution to distinguish lesional and perilesional tissue. To test this central hypothesis and overcome these challenges, we propose to: Aim 1: Identify shared mechanisms of tolerance in perilesional and post-inflammatory tissue across autoimmune diseases through a meta-analysis of suspension scRNAseq data generated from 8 autoimmune diseases across 777 patients, incorporating healed and non-affected tissue. Aim 2: Define mechanisms of tissue tolerance at the interface of inflammation in type I DM and cutaneous lupus by applying paired single nuclear sequencing (10X Genomics Flex) and ST (10X Genomics Xenium platform) to FFPE tissue from biobanked tissue (n=6 donors per disease). To support this we have developed computational frameworks for data integration and optimized generation of spatial transcriptomic data. Collectively, results from this study will enable developing frameworks to study tolerogenic mechanisms shared across autoimmune diseases that will empower the future identification of therapeutic targets and of biomarkers of tolerance that can be used to guide clinical trial development and treatment targeting and rationalization.