Human innate lymphocytes in host defense, tissue function and reproduction

The immune system is present in all tissues of the human body. In order to respond appropriately to infections, cells of the innate and adaptive immune system work in concert. In this thesis, we focused on natural killer cells (NK cells) and mucosa associated invariant T cells (MAIT cells), that are both present in peripheral blood and enriched in certain tissues such as the liver and, in case of NK cells, also the uterus where they are thought to be important regulators at the maternal-fetal interface. NK cells are able to react readily towards, among other things, virus-infected cells whereas MAIT cells are triggered by both cytokines in viral infections and bacterial metabolites during bacterial infections. Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis and is known to impact the immune system at several instances in order to establish chronic infection. Yet, with recent advances in anti- viral treatment, the infection can now be cured in a short period of time. As a result of this, it is possible to study the impact of HCV on the immune system in a dynamic setting. Therefor we investigated both NK cells and MAIT cells in HCV before, during, and after viral clearance of the infection. Despite rapid clearance of virus, imprints inflicted by chronic HCV infection remained within NK cells and MAIT cells over time. More specifically, MAIT cells were dysfunctional, present at a reduced frequency, and displayed an altered phenotype that was only partially restored upon infection clearance. Along similar lines, also the NK cell phenotype and the diversity of the NK cell compartment remained altered after rapid HCV viral clearance. We observed persistently reduced intraindividual diversity of NK cells after elimination of chronic HCV, whereas the interindividual diversity, more linked to liver damage, was restored over time. Next, we studied peritoneal MAIT cells in ascites of patients with liver cirrhosis on the basis of viral hepatitis and other causes, and in the context of spontaneous bacterial peritonitis (SBP), a frequent and severe complication occurring in these patients. First, we could recapitulate the loss of MAIT cells in peripheral blood of cirrhosis patients in line with previous studies. Of note, in ascites, MAIT cells were more frequent than in blood and displayed a tissue-residency phenotype with increased functionality as compared to peripheral blood MAIT cells. Strikingly, during SBP, MAIT cells were the most recruited immune cells to the peritoneal cavity. Lastly, we investigated uterine NK cells (uNK cells) and hypothesized that these cells underwent a stepwise differentiation in response to endometrial regeneration and pregnancy. We present a model where uNK cells continuously differentiate throughout the menstrual cycle. This differentiation was associated with a functional shift towards immunomodulation and enhanced angiogenic function, possibly to aid in spiral artery formation during pregnancy. In conclusion, we present data suggesting that chronic infections can leave a long-lasting imprint on the immune system. We further demonstrate that immune cell frequency, phenotype, and function can be altered depending on the respective tissue the cells reside in. This work increases our understanding of how innate lymphocytes respond to environmental cues, such as acute or chronic viral and bacterial challenges, as well as to normal physiological processes of the human body

Irreversible impact of chronic hepatitis C virus infection on human natural killer cell diversity

Diversity is crucial for the immune system to efficiently combat infections. Natural killer (NK) cells are innate cytotoxic lymphocytes that contribute to the control of viral infections. NK cells were for long thought to be a homogeneous population of cells. However, recent work has instead revealed NK cells to represent a highly diverse population of immune cells where a vast number of subpopulations with distinct characteristics exist across tissues. However, the degree to which a chronic viral infection affects NK cell diversity remains elusive. Hepatitis C virus (HCV) is effective in establishing chronic infection in humans. During the last years, new direct-acting antiviral drugs (DAA) have revolutionized treatment of chronic hepatitis C, enabling rapid cure in the majority of patients. This allows us to study the influence of a chronic viral infection and its subsequent elimination on the NK cell compartment with a focus on NK cell diversity. In our recent study (Nat Commun, 9:2275), we show that chronic HCV infection irreversibly impacts human NK cell repertoire diversity

Human resident liver myeloid cells protect against metabolic stress in obesity

Although multiple populations of macrophages have been described in the human liver, their function and turnover in patients with obesity at high risk of developing non-alcoholic fatty liver disease (NAFLD) and cirrhosis are currently unknown. Herein, we identify a specific human population of resident liver myeloid cells that protects against the metabolic impairment associated with obesity. By studying the turnover of liver myeloid cells in individuals undergoing liver transplantation, we find that liver myeloid cell turnover differs between humans and mice. Using single-cell techniques and flow cytometry, we determine that the proportion of the protective resident liver myeloid cells, denoted liver myeloid cells 2 (LM2), decreases during obesity. Functional validation approaches using human 2D and 3D cultures reveal that the presence of LM2 ameliorates the oxidative stress associated with obese conditions. Our study indicates that resident myeloid cells could be a therapeutic target to decrease the oxidative stress associated with NAFLD

29-Color Flow Cytometry : Unraveling Human Liver NK Cell Repertoire Diversity

Recent studies have demonstrated extraordinary diversity in peripheral blood human natural killer (NK) cells and have suggested environmental control of receptor expression patterns on distinct subsets of NK cells. However, tissue localization may influence NK cell differentiation to an even higher extent and less is known about the receptor repertoire of human tissue-resident NK cells. Advances in single-cell technologies have allowed higher resolution studies of these cells. Here, the power of high-dimensional flow cytometry was harnessed to unravel the complexity of NK cell repertoire diversity in liver since recent studies had indicated high heterogeneity within liver NK cells. A 29-color flow cytometry panel allowing simultaneous measurement of surface tissue-residency markers, activating and inhibitory receptors, differentiation markers, chemokine receptors, and transcription factors was established. This panel was applied to lymphocytes across three tissues (liver, peripheral blood, and tonsil) with different distribution of distinct NK cell subsets. Dimensionality reduction of this data ordered events according to their lineage, rather than tissue of origin. Notably, narrowing the scope of the analysis to the NK cell lineage in liver and peripheral blood separated subsets according to tissue, enabling phenotypic characterization of NK cell subpopulations in individual tissues. Such dimensionality reduction, coupled with a clustering algorithm, identified CD49e as the preferred marker for future studies of liver-resident NK cell subsets. We present a robust approach for diversity profiling of tissue-resident NK cells that can be applied in various homeostatic and pathological conditions such as reproduction, infection, and cancer

Chronic hepatitis C virus infection irreversibly impacts human natural killer cell repertoire diversity.

Diversity is a central requirement for the immune system's capacity to adequately clear a variety of different infections. As such, natural killer (NK) cells represent a highly diverse population of innate lymphocytes important in the early response against viruses. Yet, the extent to which a chronic pathogen affects NK cell diversity is largely unknown. Here we study NK cell functional diversification in chronic hepatitis C virus (HCV) infection. High-dimensional flow cytometer assays combined with stochastic neighbor embedding analysis reveal that chronic HCV infection induces functional imprinting on human NK cells that is largely irreversible and persists long after successful interventional clearance of the virus. Furthermore, HCV infection increases inter-individual, but decreases intra-individual, NK cell diversity. Taken together, our results provide insights into how the history of infections affects human NK cell diversity

MAIT cells are enriched and highly functional in ascites of patients with decompensated liver cirrhosis.

Patients with advanced liver cirrhosis have an increased susceptibility to infections. As part of the cirrhosis-associated immune dysfunction, mucosal associated invariant T (MAIT) cells, that have the capacity to respond towards bacteria, are severely diminished in circulation and liver tissue. However, MAIT cell presence and function in the peritoneal cavity, a common anatomical site for infections in cirrhosis, remain elusive. To study this, matched peripheral blood and ascites fluid were collected from 35 patients with decompensated cirrhosis, with or without spontaneous bacterial peritonitis (SBP). MAIT cell phenotype and function were analyzed using high-dimensional flow cytometry and obtained data was compared to blood samples of healthy controls (n=24) and patients with compensated cirrhosis (n=11). We found circulating MAIT cells to be severely decreased in cirrhotic patients as compared to controls. In contrast, in ascites fluid, MAIT cells were significantly increased together with CD14+ CD16+ monocytes, ILCs, and NK cells. This was paralleled by elevated levels of several pro-inflammatory cytokines and chemokines in ascites fluid as compared to plasma. Peritoneal MAIT cells displayed an activated tissue-resident phenotype and this was corroborated by increased functional responses following stimulation with E. coli or lL-12 + IL-18 as compared to circulating MAIT cells. During SBP, peritoneal MAIT cell frequencies increased most among all major immune cell subsets, suggestive of active homing of MAIT cells to the site of infection. CONCLUSIONS: Despite severely diminished MAIT cell numbers and impaired phenotype in circulation, peritoneal MAIT cells remain abundant, activated, and highly functional in decompensated cirrhosis and are further enriched in SBP. This suggests that peritoneal MAIT cells could be of interest for immune intervention strategies in patients with decompensated liver cirrhosis and SBP