HCV infection is associated with immune dysregulation and B cell Non-Hodgkins lymphoma (HCV-NHL). We have previously shown in vitro that HCV core protein differentially regulates T and B cell functions through two negative signaling pathways, programmed death-1 (PD-1) and suppressor of cytokine signaling-1 (SOCS-1). In this report, we performed a detailed immunologic analysis of T and B cell functions in the setting of HCV-NHL. We observed that T cells isolated from patients with HCV-NHL exhibited an exhausted phenotype including decreased expression of viral-specific and non-specific activation markers; whereas B cells exhibited an activated phenotype including over-expression of cell activation markers and immunoglobulins compared to healthy subjects. Individuals with HCV alone or NHL alone exhibited abnormal T and B cell phenotypes, but to a lesser extent compared to HCV-NHL. This differential activation of T and B lymphocytes was inversely associated with the expression of PD-1 and SOCS-1. Interestingly, blocking PD-1 during TCR activation inhibited SOCS-1 gene expression, suggesting that these regulatory pathways are linked in T cells. Importantly, blocking PD-1 also restored the impaired T cell functions observed in the setting of HCV-NHL. These results support a coordinated mechanism by which HCV might cause immune dysregulation that is associated to HCV-NHL.

<p>We are all born germ-free. Soon after birth, microbes colonize our body’s surfaces, with the intestine housing the highest density of microbes on earth. Most of us remain blissfully unaware of this co-existence because inflammatory responses to the indigenous microbes are normally not triggered. Nonetheless, intestinal microbes are true educators of our immune system, which is exemplified by the immature immune system observed in germ-free animals. Accumulating evidence suggests that microbial exposure and/or composition impacts on immune regulation. As an example, isotype switch to immunoglobulin E (IgE) is normally very tightly regulated such that in healthy individuals and mice, serum levels are maintained at very low levels. In contrast, total serum IgE levels are elevated in germ-free mice, indicating that in the absence of microbes the regulatory pathway that maintains IgE at basal levels is disrupted. We hypothesize that in the absence of stimuli from the resident intestinal bacteria the immune system does not receive adequate educational signals. We showed that in germ-free mice class switch recombination (CSR) to IgE occurred at intestinal mucosal lymphoid sites a few weeks after birth. IgE levels then remained at elevated levels throughout life, even when intestinal bacteria were introduced after weaning. In the first part of this thesis, the mechanisms involved in this hygiene-induced IgE were investigateted. In a second part, the immunoregulatory role of commensal bacteria was extended to a model of autoimmunity.</p> <p>Collectively these results demonstrate a new dimension of the impact of intestinal symbionts on the immune system: they dictate baseline immune system regulation. Elucidating the mechanisms whereby microbes induce immunoregulatory pathways may give insights into the increasing prevalence of allergic- and autoimmune diseases.</p> / Doctor of Philosophy (PhD)

Bc. Klára Horáčková DIPLOMA THESIS Mechanisms of immune dysregulation leading to inflammatory bowel disease Abstract Inflammatory bowel disease (IBD) is a complex disorder characterized by chronic inflammation of the gastrointestinal tract. Classical IBD is a multifactorial disease with adulthood or later-childhood onset. However, children with very early onset IBD (VEO-IBD, before 6 years of age) are a specific cohort, whose pathology can be caused by severe genetic defects in genes connected to immune homeostasis in the gut. We aimed to identify the causal genetic variants in 20 pediatric patients diagnosed with IBD (age of onset from 3 to 154 months) using whole exome sequencing (WES). We evaluated several bioinformatical approaches for WES data analysis. This included a comparison of two methods of variant identification using VarScan2 or GATK4-based tools. Furthermore, we compared 4 gene lists ("virtual panels") for variant filtering, one of which was compiled purposefully for this thesis. We identified and validated via segregation analysis 5 causal variants in 4 genes (DUOX2 compound heterozygote, FOXP3, NLRP3 and NOD2) accounting for 20 % of the cohort. NOD2 (p.A755V) variant has already been reported in IBD cases, while DUOX2 (p.R1216W + p.A1131T), FOXP3 (p.H400L) and NLRP3 (p.V200M) were newly...