Exploring T-cell heterogeneity in Chornic Lymphocytic Leukemia to unravel immune-based therapeutic strategies.

Chronic Lymphocytic Leukemia (CLL) is the most prevalent haematological malignancy among adults in the western world and, unfortunately, remains a deadly and incurable disease. CLL is characterized by the chronic accumulation of abnormal B-lymphocytes that circulate between the peripheral blood and lymphoid organs of patients. The clinical management of CLL has advanced significantly in the last few decades, with the introduction of targeted therapies representing a milestone for the treatment of patients with advanced and aggressive disease. Nevertheless, the lack of an established curative approach in standard clinical practice leads to the need for lifelong treatments, associated with a strong emotional and physical burden for CLL patients. Moreover, CLL can progress into more aggressive malignancies with highly unfavourable outcomes, including but not restricted to Richter Syndrome (RS). As a result, there is an urgent need for the development of effective strategies to treat high-risk CLL patients.
The elucidation of the pathobiological features driving malignant transformation and progression is a key requirement to identify novel targets with translational potential. A prominent and well-established trait of CLL cells is their extraordinarily high dependence on complex interactions with non-malignant surrounding cells for survival and proliferation. Interestingly, the cellular interplay driving CLL progression is not constant and occurs in a unique niche located within affected lymph nodes, which are consequently referred to as the reservoirs of CLL. A key component of the lymph node tumour microenvironment (TME) is the T-cell compartment, often regarded as the main force in anti-tumour immunity. Even though T-cells in the leukemic niche can initially identify the malignant clone and start an adaptive response against the tumour, they eventually become anergic and dysfunctional. This phenomenon, known as T-cell exhaustion, is driven by several mechanisms, including but not limited to persistent antigen exposure and regulatory signals within the tumour microenvironment (TME). The anatomical location of the CLL proliferative niche and the consequent infiltration of T-cells within the lymph node TME establish this malignancy as an optimal candidate for benefiting from immunotherapeutic approaches that halt the regulatory mechanisms preventing T-cell anti-tumour immunity.
The aim of this thesis is to identify and functionally characterize novel therapeutic approaches to restore T-cell-mediated anti-tumour immunity in CLL patients. For this purpose, the following manuscript is divided into five chapters, each supported by a publication that covers a different stage of the pre-clinical research journey. The first chapter describes the pre-clinical models available in CLL research, focusing on the Eµ-TCL1 transgenic mice, the most established pre-clinical approach for the study of CLL. Alongside this chapter, the benefits and pitfalls of the most popular models are highlighted, as well as the challenges currently faced in the development of patient-derived xenograft (PDX) models for CLL. The second chapter revolves around mass cytometry (MC), a novel technique used for the phenotypic characterization of single cells with unprecedented resolution. In this publication, we draw from our experience to describe a protocol adapted for the use of MC in the exploration of the TME in CLL. The third chapter covers a large collaborative study focused on comprehensively characterizing the T-cell landscape across various lymphoid compartments in CLL patients using a state-of-the-art multi-omics approach. This publication, currently under submission, has the objective to dissect T-cell dysfunction in CLL and pinpoint promising and targetable markers that could restore T cell function and, ultimately, an effective anti-tumour immune response against CLL cells. Importantly, we identify galectin-9 (Gal-9) as a potential target for this purpose, and successfully delay leukemic development through Gal-9 inhibition in a pre-clinical CLL mouse model. Additionally, we identify the enrichment of Tr-1-like cells in CLL patients, a CD4+ T cell subset reported to have anti-tumour functions through direct cytotoxic mechanisms upon reactivation in a range of malignancies. In the fourth chapter, we further explore the role of the previously identified cytotoxic CD4+ T cells in CLL development and progression. Of particular relevance, we show that donor-derived CD4+ T cells activated in vitro are able to control the development of an aggressive CLL cell line in an immunodeficient humanized mouse model. The final chapter addresses the importance of cytokine biology in CLL pathogenesis, focusing on the role of interleukin-27 (IL-27) in CLL progression. Throughout this publication, we use transgenic mouse models and patient samples to demonstrate the strong anti-tumour properties of this cytokine in CLL development in a T-cell-mediated mechanism. Moreover, we identify several cellular markers involved in the aforementioned process.
Overall, this thesis aims to provide a comprehensive characterization of the complex T-cell landscape in CLL. The insights gained from this analysis are then applied in a pre-clinical setting to test potential novel therapeutic approaches aimed at restoring T-cell-mediated anti-tumour immunity in CLL patients. Key therapeutic approaches identified include the blockade of galectin-9, the reactivation of cytotoxic CD4+ T cells, and the administration of IL-27, all of which successfully controlled leukemic development in a pre-clinical setting. Further research into the mechanisms responsible for these observations must be performed in order to assess their feasibility for clinical use in CLL patients.