Systemic sclerosis (SSc) is a complex autoimmune connective tissue disorder. SSc presents with severe pathological clinical manifestations, including vasculature abnormalities, dysregulation of the immune system, and excessive extracellular matrix deposition that results in tissue fibrosis. How immune system abnormalities impact SSc remains poorly understood. Here we sought to explore the role of co-inhibitory receptors (co-IRs), which are important regulators of autoimmune responses. Previous studies showed altered co-IR expression in various autoimmune diseases, including SSc. Here we show that T cells co-expressing the co-IRs programmed cell death protein 1 (PD-1) and T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) are expanded in lung tissue obtained from SSc patients, as compared to healthy controls (HC). Furthermore, we found a significant association between the frequency of PD-1+TIGIT+ CD4+ T cells and lung disease in SSc patients. In addition, PD-1+TIGIT- and PD-1+TIGIT+ CD4+ cells in SSc patients showed an altered balance in cytokine production, characterized by reduced secretion of Interferon-γ, a cytokine with known anti-fibrotic properties, and increased levels of Interleukin-4, which is known for its pro-fibrotic activities. To test the impact of this changed cytokine balance on fibroblast biology, we co-cultured PD-1+TIGIT- and PD-1+TIGIT+ CD4+ T cells with normal dermal fibroblasts and found that PD-1+TIGIT- and PD-1+TIGIT+ T cells from SSc patients showed a reduced capacity to suppress collagen production, compared to the same subsets from HC subjects. Thus, co-IR-expressing T cells from SSc patients show features of dysfunction and may have lost anti-fibrotic activities. To further define the phenotype and functions of co-IR-expressing T cells subsets in SSc patients, we next designed a comprehensive immunophenotyping panel for full spectrum flow cytometry (FSFC) that included detection of lineage-defining transcription factors. Using this novel panel and an unbiased analysis approach, we compared T cell subset composition in peripheral blood mononuclear cells from HC subjects, and SSc and systemic lupus erythematosus (SLE) patients. Our analysis revealed broad shifts such as a decrease in the naïve CD4+ and CD8+ T cell compartment in SSc and in SLE patients. Importantly, changes in specific T cell subsets that were discovered in SLE patients, but not SSc patients, had a broad increase in T helper (Th)1 and T cytotoxic (Tc)1 subsets and a decrease in Th2/Tc2 subsets compared to HC subjects. Interestingly, we found a distinct Tc1 subset with exhaustion characteristics that was significantly reduced in both SSc and SLE patients compared to HC subjects. In the γδ T cell population, we found that while T-bet+ Vδ2 cells were decreased in SSc and SLE patients, the T-bet+ Vδ1 subset showed proliferative characteristics and was increased in SLE. Importantly, our analysis revealed differences in specific T cell subsets between SSc patients treated with immunosuppressants vs untreated patients, including an increase in Th17 cells in diffuse cutaneous SSc (dcSSc) patients that were not treated with immunosuppressants, and an increase in memory regulatory T cells in both dcSSc and limited cutaneous SSc (lcSSc) patients that were not treated with immunosuppressants. Our study demonstrates the value of a multiparameter FSFC panel in the identification of differentially represented and novel subsets of T cells in SSc and other autoimmune diseases. We demonstrated that T cell subset composition is altered in the peripheral blood of SSc patients and show that features of specific T cell subsets’ dysfunction are potentially contributing to SSc pathophysiology.

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<p>An acute bout of exercise causes short-term changes in the immune system in both children and adults. It has been well-established that exercise induces an inflammatory response. Especially in children, cytokines play an important role in balancing anabolic and catabolic processes of growth. Existing evidence suggests cross-talk between inflammation and oxidative stress. Reactive oxygen species are also found to transiently increase in response to exercise, affecting muscle adaptation post-exercise. Characterizing the exercise-induced inflammatory and oxidative stress responses in children compared to adults will start clarifying the transition from the child phenotype to that of an adult. Ten children aged 8-10 and 12 adults aged 19-21 performed 2×30min bouts of continuous cycling, separated by a 6min rest period, at a target work rate of 60% of their maximum aerobic capacity. Blood samples were collected pre-exercise and immediately post-exercise, and analyzed for<strong> </strong>neutrophil count, systemic oxidative and inflammatory markers (tumor necrosis factor alpha, interleukin 6, protein carbonyls, malondialdehyde, elastase), intracellular neutrophil-derived reactive oxygen species (using 3 fluorescent markers detected by flow cytometry), and <em>in vitro</em> production of neutrophil-derived myeloperoxidase and interleukin 8. Compared to the post-exercise increase in absolute neutrophils in men, boys showed no change. However, intracellular neutrophil reactive oxygen species production increased for boys and not for men. Boys also demonstrated higher overall protein carbonyl levels, whereas men showed higher overall malondialdehyde. Both boys and men showed a positive correlation between tumor necrosis factor alpha and elastase, with a steeper slope seen in boys. Although there were other correlations observed in boys and men, no others existed in both. The differences observed in the exercise-induced inflammatory and oxidative stress response may indicate growth-mediated adaptive responses to exercise during childhood development.</p> / Master of Science (MSc)