Innate lymphoid cell plasticity and heterogeneity in human tissues

Abstract: The human immune system is a vital mechanism that protects the host from outside threats. The two main branches of this system, consisting of innate and adaptive immunity, aid the host when dangers of both imminent and protracted nature occur. Innate lymphoid cells (ILC) play a key role in innate immunity and are classified into distinct groups based on their function, transcriptional profile and development. Their discovery is young, with much research needed to understand all aspects of their phenotype, genotype, behavior in homeostasis and disease. This thesis describes the breakthroughs my collaborators and I were able to reach during my five years of doctoral studies, in order to better understand the biology and physiology of ILC. In the first part of this thesis I describe the history and classification of ILC, the efforts being done so far by the field to understand their development, and the possible combinations of changes in ILC status, known as plasticity or trans-differentiation properties of ILC under particular environments or stimuli. As Paper II’s results are based on a cohort of Inflammatory Bowel Disease (IBD) samples, and in Paper I we analyze ILC in intestinal biopsies from IBD patients, I also outline the main characteristics representing this disorder while focusing on the role of ILC in IBD. Next, after defining the aim of my studies, I describe how the data was obtained, as a big part of my work consisted in learning how to handle methods and technologies such as flow cytometry, fluorescence-activated cell sorting (FACS) and single cell RNA sequencing, among others. Finally, a discussion of the results is aimed at highlighting the major breakthroughs achieved. In Paper I, we set out to better understand the function of the Ikaros family of transcription factors in ILC, focusing our efforts on IKZF3 (encoding Aiolos) and its role in ILC trans- differentiation via a drug-induced silencing approach with the immune-modulatory agent lenalidomide. In Paper II, a large cohort of IBD samples was analyzed in order to find disturbances in peripheral blood ILC protein expression. We were able to uncover differences in several activation proteins in the IBD cohort, when compared to a like-sized cohort of samples from healthy controls. In Paper III, a big effort was put into implementing Smart- seq2 RNA sequencing technology to a large number of ILC from a variety of tissues. This allowed us to better understand the heterogeneity of ILC in the circulation, secondary lymphoid and mucosal tissues. We generated a large dataset that will require time to be exploited in full, constituting a roadmap for future studies aimed at understanding human ILC biology and function. In summary, the work presented in this thesis provides findings and datasets that have the potential to advance the ILC field.