T-LGL leukemic cells are believed to arise from healthy control counterparts in the peripheral blood that display the classical profile of an antigen-experience effector cell. T-LGL leukemia forms a spectrum of proliferations ranging from oligoclonal to monoclonal and from asymptomatic to clinically malignant. T-LGL cells are observed in many different disease contexts and may therefore be triggered by a vast amount of different antigens. Some, but not all, T-LGL leukemic cells present with (epi)genetic aberrancies. From these, mutations in the SH2 domain of the STAT3 gene are thought to be most important in disease pathogenesis (10), even though only 40-60% of T-LGL patient cells harbor this mutation. In this thesis we aimed to elucidate if T-LGL cells present in the spectrum of proliferations are causal for the associated diseases, or arise as a consequence. In addition, we aimed to establish at the molecular and functional levels, what discriminates malignant T-LGL cells from their healthy control reactive counterparts. In Chapter 1, general background information on T-LGL leukemia disease etiology and relevant common immunology is given. In Chapters 2 and 3 we addressed the spectrum of T-LGL proliferations and signatures in the TRB repertoire using next generation sequencing (NGS) technology. Chapter 2 is a detailed description of the next generation sequencing pipeline developed during the COVID-19 pandemic that can be utilized to explore the TRB repertoire in health and disease and to assess antigen specificity. This approach was validated on a COVID-19 patient cohort. A clustering algorithm was applied to group highly similar TRB CDR3 amino acid sequences. The public part of the TRB repertoire was filtered out by removing TRB sequences associated with common diseases such as Influenza, EBV and CMV. Cross-referencing of the remaining sequences with a publicly available dataset of TRB sequences mapped to the SARS-CoV-2 genome showed that clusters associated with critical outcome more often target antigens of nonstructural proteins of ORF1a/b. This implies that T-lymphocyte reactivity towards peptides from these proteins may negatively affect disease severity. In Chapter 3 we searched for associations between TRB gene repertoire patterns and clinical manifestations with the use of the validated pipeline described in Chapter 2 in order to discriminate between T-LGL proliferations developing in different clinical contexts and/or displaying distinct clinical presentations. The combination of longitudinal analyses, creation of connectivity networks and combinatorial pattern discovery analysis showed that the TRB gene repertoire of patients with T-LGL proliferations is context-dependent, displaying distinct clonal architectures in different disease contexts and revealed clear signs of (auto)antigenic stimulation. The data further showed that T-LGL proliferations form a disease spectrum, with the more monoclonal populations giving rise to neutropenia and other symptoms on the one end and the more reactive oligoclonal proliferations on the other end. Finally, longitudinal analyses revealed that T-LGL cells showed profound temporal clonal dynamics and might occur as an epiphenomenon, possibly reflecting cell populations that are reactive towards tumor antigens when co-occurring with other malignancies. Chapter 4 provides an in depth overview of a novel mechanism of STAT3 and ERK1/2 activation in T-LGL leukemia. By integrating mRNA and miRNA sequencing datasets, we revealed that miR-181a is involved in the regulation of pSTAT3 and pERK1/2 by inhibiting the respective targets SOCS3 and DUSP6. We established that miR-181a inhibition in T-LGL patient cells restores SOCS3 and DUSP6 protein expression. Furthermore, in the TL1 cell line, which is a model cell line for T-LGL leukemia, we could show that miR-181a acts centrally in T-LGL leukemia, driving STAT3 and ERK1/2 phosphorylation by SOCS3 and DUSP6 inhibition, eventually leading to lower sensitivity to FAS-mediated apoptosis. Chapter 5 describes phenotypical characteristics of T-LGL cells in the light of exhaustion and shows that the functional aberrancies that T-LGL cells display might indeed be related to exhaustion. T-LGL cells show phenotypical characteristics of exhaustion as PD1, CD244 and CD57 are all higher expressed in these cells. On top of that, T-LGL cells are functionally impaired, not being able to produce cytokines such as TNFα and IFNγ and showing less membrane expression of CD107a, which is a marker for degranulation. We observed that T-LGL cells have an altered proliferative potential that is arguably caused by defective mTOR signaling since pAkt, pS6 and p4E-BP1 signaling was diminished upon TCR stimulation in T-LGL cells. Ultimately, in the General Discussion in Chapter 6, an integrated perspective is given based on the data presented in the chapters 2-5 of the thesis and in combination with data reported in literature future research studies are discussed. In future studies emphasis should be put on antigenic specificity of T-LGL cells through complex tools that predict pMHC/TR interactions and eventually patient specific stimulations with pools of predicted peptides. In addition, molecular heterogeneity of T LGL leukemic cells with defined TR specificity at the single-cell level should be evaluated with regard to potentially targetable pathways, since therapy for T LGL patients has remained unchanged for decades. Such single-cell level approaches should also be utilized in order to further evaluate relevant pathways and biological processes that characterize cells with a certain TR specificity and to advance understanding of T LGL leukemia heterogeneity.