Comprehensive characterization of gene expression response to type-I and type-II interferon in healthy and diseased conditions

Interferons (IFNs) are critical regulators of the immune system, with special importance for viral infections, autoimmune diseases, and cancer. However, there are challenges in distinguishing the effects of different IFN types and understanding their cell-type-specific responses. Single-cell sequencing can address these gaps but requires robust tools for cell-type classification and precise analysis of IFN-mediated effects. To better characterize IFN- and cell-type-specific gene expression responses, novel bioinformatics approaches are needed. One of the main challenges in single-cell analysis is cell-type classification. Most approaches use expression information of all expressed genes to provide cell type labels. However, they often introduce bias into statistical procedures when testing the same genes for differential expression that were already used for cell typing. In the first chapter of my thesis, I addressed this issue by using only small sets of robust immune cell-type-specific genes for cell typing within a random forest model. While most studies have generated such gene expression signatures (GESs) from single gene expression datasets, I developed a novel GES discovery workflow based on similarities in gene expression across seven single-cell cancer datasets. Compared to existing algorithms and published GESs, my approach showed superior or comparable performance, significantly improved the classification of myeloid cells and enhanced downstream analysis of peripheral blood mononuclear cell (PBMC) datasets. Thereby, I establish an unbiased method for classifying immune cells and statistical investigation of expression differences between cell types. In the second part, I dissected distinct effects of IFN-I and IFN-II across different cellular, experimental, and disease contexts. Published IFN GESs have been purely generated from only single gene expression datasets within specific cellular contexts and primarily comprise genes induced by IFN-I. I used five different bulk tissue RNA-sequencing datasets of IFN stimulation and applied a novel meta-analysis workflow to resolve GESs with specificity for IFN-I and IFN-II response. My IFN GESs had greater functional relevance to IFN-type-specific response and higher coherence than most published signatures. My IFN-II GES detected IFN- II response of not only myeloid cells but also B cells, hematopoietic cells, and naïve T cells. Further, I demonstrated the relevance of IFN-I GES in disease severity of lupus nephritis, and IFN-II GES as predictive biomarker for immune checkpoint inhibitor response. I provide a more precise distinction between IFN-I and IFN-II responses at the cell type level, as well as their relevance to disease progression and therapy outcomes. In the final part, I characterized immune cell-type-specific responses to IFNs as comprehensive and objective studies of these responses are still lacking. Previous studies do not provide cellular or temporal resolution, nor compare responses across different IFNs within a single study. To address this gap, I applied the tool sets established in Chapter 1 and 2 on a novel temporal CITE-Seq dataset of IFN-I and IFN-II stimulation of human PBMCs. I showed that all immune cell types exhibited transient responses to IFN-I stimulation while only myeloid and B cells responded to IFN-II, with distinct dynamic patterns. Furthermore, I identified five unique temporal gene groups specific to monocyte responses to IFN-I or IFN-II. Those groups consist of genes that play key roles in distinct immunological pathways. My findings enable a more detailed characterization of IFN-mediated responses in distinct immune cell populations compared to those provided by published datasets or GESs. In this thesis, I introduce novel bioinformatics tools that address key challenges in immune cell classification and the study of IFN responses. These methods advance the resolution of immune profiling in single-cell data and provide more precise insights into IFN-mediated immune cell type responses in both health and disease. My approaches overcome the limitations of existing workflows, offering new insights into IFN biology and its relevance to disease mechanisms, as well as potential applications in biomarker research and therapy.