Ramon Klein Geltink

CD8 T cells are a part of the adaptive immune system and are capable of eliminating cancer cells. It is well established that CD8 T cell function can be regulated by T cell metabolism. Upon activation, T cells increase the breakdown of glucose (glycolysis) and increase mitochondrial metabolism. The increase in glycolysis promotes rapid energy production and increases the levels of glycolytic intermediates to be used for nucleotide, amino acid, and lipid synthesis. T cells that have a high glycolytic rate also have increased expression of pro-inflammatory cytokines: interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα). In addition, exogenous pyruvate in other cell types has been associated with increased glycolysis. In light of this, we were interested in determining if extracellular availability of pyruvate could improve CD8 T cell anti-tumor function. We hypothesize that supplementing CD8 T cells with sodium pyruvate (NaPyr) will improve CD8 T cell ability to sustain the production of cytokines and cytotoxins to kill cancer cells. We found that CD8 T cells restimulated in the presence of exogenous pyruvate had increased expression of IFNγ, TNFα, and perforin compared to NaCl-exposed T cells due to differences in posttranscriptional regulation. In addition, CD8 T cells exposed to NaPyr had improved tumour cell killing. We then mechanistically explored how exogenous pyruvate improves T cell function. We found that NaPyr supplemented CD8 T cells had an increase pyruvate uptake and a decrease in glucose uptake compared to NaCl-exposed CD8 T cells. We then performed GC-MS on CD8 T cells cultured in labelled or unlabelled glucose to assess how NaPyr supplementation alters metabolite levels, as well as glucose metabolism. We found that NaPyr supplementation increases levels of 3-phosphoglycerate and phosphoenolpyruvate, TCA cycle metabolites, and many amino acids. We used activators/inhibitors of metabolic enzymes/transporters to determine if specific metabolic node(s) were important for the NaPyr supplementation phenotype. We found some interesting observations but were unable to determine a specific mechanism.Determining how extracellular pyruvate alters metabolism could provide insight into metabolic pathways that may be important for CD8 T cell function and potentially use this knowledge to improve immunotherapies against cancer.

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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.

Macrophages are multifunctional innate immune cells that can promote or dampen inflammation. Various stimuli including cytokines and pathogen- or damage-associated molecular patterns activate macrophages, reprogramming them into a diverse array of possible subtypes. The signals guiding the differing functional states of macrophages are of particular interest in the search to provide improved treatment of diseases in which macrophage-mediated inflammation is implicated. During macrophage activation, metabolic reprogramming is crucial to support various pro- and anti-inflammatory activation states. Xanthine oxidoreductase (XOR) is a metabolic enzyme which catalyzes the final two steps in purine catabolism, producing uric acid from hypoxanthine and xanthine. XOR activity has previously been shown to support pro-inflammatory IL-1β production in macrophages, however the broader role of XOR activity on macrophage activation remains to be fully elucidated. We therefore aimed to investigate the role of XOR activity on macrophage activation and function.Bone marrow-derived macrophages were generated from adult C57BL/6 mice and using LPS(+IFNγ) or IL-4 in the presence of the XOR inhibitor febuxostat. Global metabolic profiles were assessed using the Seahorse assay, which uses proxies to measure mitochondrial and glycolytic metabolism. Expression of key markers of macrophage activation were assessed by flow cytometry. Levels of signaling mediators for Toll-like receptor 4 (TLR4, which recognizes LPS), components of LPS-induced pro-inflammatory complex NLRP3 inflammasome, and its product IL-1β, were measured by immunoblotting.We found that inhibition of XOR resulted in a preservation of mitochondrial respiratory function in LPS+IFNγ-stimulated macrophages, which are known to progressively lose this function. XOR inhibition resulted in blunted upregulation of canonical markers of activation in both LPS+IFNγ- and IL-4-stimulated macrophages. LPS-stimulated macrophages contained lower levels of IL-1β when XOR was inhibited, consistent with observed changes in TLR4 signaling mediators, including a decrease in the adaptor protein MyD88, and differential modulation of mitogen-activated protein kinase activity.XOR activity appears to support both LPS(+IFNγ)- and IL-4-induced macrophage activation. Further elucidation of the mechanism linking purine catabolism and macrophage activation could uncover novel targets for the treatment of diseases in which macrophages are implicated.

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