Ramon Klein Geltink

Adoptive T cell therapy (ACT) is a promising immunotherapeutic strategy, often resulting in complete remission in blood cancers. However, a major hurdle with the effectiveness of ACT in solid tumours is the hostile tumour microenvironment (TME). Challenges linked to TMEs include metabolic competition between immune and tumour cells, lack of tumour infiltration, and poor survival of infiltrated cells. Previous research has shown that transiently restricting glucose in fully activated CD8+ effector T cells in vitro reprograms cellular metabolism, and significantly upregulates protein expression of the glucose transporter Slc2a1/Glut1 without altering Glut1 mRNA expression. Further, these Glut1hi CD8+ T cells showed drastically improved tumour-killing ability upon reinfusion in vivo: however, the mechanistic underpinnings of the regulation of Glut1 and the effects of this high Glut1 expression remain unclear. We therefore sought to determine the role of Glut1 activity in CD8+ T cell metabolic reprogramming, and whether this high Glut1 expression during the ACT preconditioning phase mediates better tumour killing in vivo. We found that genetic deletion of Glut1 or transient blocking with a Glut1-specific reversible exofacial inhibitor (ethylidene glucose) 72 hours after activation of CD8+ T cells blunted glycolytic reserve and the oxidized subcellular redox state, both metabolic fitness characteristics conferred by transient glucose restriction (TGR). Transient Glut1-inhibition in vitro also blunted in vivo anti-tumour function of TGR Glut1hi CD8+ T cells, despite the use of a reversible inhibitor of Glut1. We found decreased donor-derived CD8+ T cells in blood, tumour, and spleen, and decreased cytokine production in tumour infiltrating lymphocytes. This suggests that Glut1 non-transcriptional upregulation and in vitro activity confers a long-lasting benefit for ACT efficacy in vivo. Mechanistically, we observed changes in glucose carbon allocation, but not basal glycolysis due to loss of Glut1 activity, which could underlie the benefits of Gluthi CD8+ T cells. Taken together, increased Glut1 expression in fully activated CD8+ cells can augment the ability of donor CD8+ T cells to clear tumours in pre-clinical models, suggesting that Glut1 plays a vital role in CD8+ T cell adoptive therapy.

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The human immune system relies on the function and balance of various immune cell subsets and their interactions. Immune cells undergo a series of differentiation steps following a lineage-tree structure stemming from hematopoietic stem cells to reach their mature cell state. During differentiation of immune cells in both homeostasis and pathological processes, many cellular features, including gene expression patterns, are shared by fully differentiated immune cell sub-types. The process of immune cell differentiation is complex and not fully understood. Additionally, aberrant function and balance plays a major contributing role in the pathogenesis of many immunological disorders, including systemic lupus erythematosus.In this thesis, I propose LaRCH, a tree-structured neural topic model as a method to quantitatively characterize shared hierarchical features between cell subsets. In this model, single-cell gene expression profiles are represented by a mixture of topics consisting of latent features that follow an underlying tree structure, mirroring the dynamics of cellular differentiation.I present findings of our model trained on simulated single-cell RNA sequencing based on cell-sorted bulk RNA-seq data and a scRNA-seq dataset of over 1.2 million cells from individuals with variable lupus disease phenotypes. The cellular topic profiles estimated by our model markedly improve cell type deconvolution accuracy over traditional methods. Trained model parameters of LaRCH illustrate cell-type specific transcriptomic differences between SLE phenotypes, revealing the contributions of multiple immune cell types in the manifestations of lupus. I also identify a number of candidate genes that may have implications in the driving mechanisms that contribute to lupus disease pathogenesis. Ultimately, LaRCH is able to capture the hierarchical context between immune cell subsets by simultaneously identifying shared and distinct latent features amongst cell subtypes within heterogeneous cell samples.

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No abstract available.

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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