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Fleur S. Peters et al.
CLL T-cells, it might explain why higher levels of HIF1α levels do not coincide with increased glycolysis in CD8+ T cells from CLL patients.25 These cells also show reduced expression of PGC1α and impaired mitochondrial biogen- esis upon T-cell activation.25 Whether PGC1α plays a causal role in the observed dysfunction or is a conse- quence of increased PD-1 expression remains unclear, however overexpression of PGC1α could be a strategy to improve the metabolic fitness of CAR T cells.91
Besides inducing elevated levels of ROS, hypoxia also mediates immunosuppression through induction of CD39 and CD73, two ectonucleotidases that metabolize ATP to adenosine. In the CLL TME, high adenosine levels are detected and CLL cells express CD39 and CD73.98 Serra et al.99 showed an anti-apoptotic effect of adenosine on CLL cells, whilst the effect of adenosine on T cells was 2-fold, firstly it reduced IFNγ production and proliferation of effector CD8+ T cells and it then induced proliferation of Tregs.100 The adenosine signaling axis has not been studied yet in T cells of CLL patients, and we hypothesize that it could play an important role. This is strengthened by a study in the TCL1 mouse model. Targeting the A2A extra- cellular adenosine receptor led to restoration of immune
Table 1. Epigenetic mechanisms.
competence towards the CLL cells by increased degranu- lation and cytokine release, and decreased Treg expansion.101 These data indicate the potential of targeting the adenosinergic axis for immunotherapeutic approaches in CLL.
Taken together, the epigenetic profiles of CD4+ and CD8+ T cells can demonstrate the differentiation they have undergone which will be helpful in determining the level of exhaustion and potential reversibility of the dys- functional phenotype of CLL T cells. In addition, metabol- ic alterations in the CLL TME likely cause T-cell aberra- tions through epigenetic mechanisms.
Future considerations
There are several outstanding questions in the field of CLL-induced T-cell dysfunction (Table 2) and given the rapid development of technologies available to study the epigenome, the possibilities seem ample. Study design will be critical to permit accurate interpretation and inte- gration with large multi-omics datasets. It is essential to choose the correct ‘normal’ counterpart for comparison; in
Gene expression, and by extension cellular differentiation and function, is tightly regulated by epigenetic mechanisms. These are reversible, chemical mod- ifications on top of the genomic information encoded in the DNA, without altering the underlying sequence. In general, DNA methylation, histone modifi- cations and 3-dimensional (3D) chromatin folding are identified as epigenetic mechanisms. Together these mechanisms dictate gene expression in response to environmental cues, cellular context and as instructed by underlying genomic sequences.
Histone modifications
Histone proteins form the structure, a nucleosome, around which the DNA double helix is wound. This nucleosome consists of two copies of each core histone: H2A, H2B, H3 and H4, each of which can have a vast amount of post-translational modifications (PTM).106 PTM on histones include acetylation, methylation, phosphorylation, sumoylation and ubiquitylation and are present on different locations on the histone tail. Although for some PTM the effect on gene expression is unknown, there are several well-studied histone modification marks that allow for functional annotation of a certain genomic loca- tion. H3K4me3, H3K2me1, H3K36me3, H3K27ac and H3K9ac are associated with active gene regions such as promoters, enhancers and intergenic regions; H3K27me3 and H3K9me3 are associated with silenced chromatin.106 Acetylation of histones is regulated by enzymes called histone acetyltransferases and histone deacetylases and histone methylation by histone methyltransferases (HTM) and histone demethylases.
Table 2. Outstanding questions.
- Is recovery of T-cell function and phenotype after CLL treatment due to plasticity and/or selective survival of functional T -cells?
-To which extend are the CD4+ T cells tumor supportive in CLL and could they also provide anti-tumor immunity in CLL?
-What is the level of exhaustion of CLL T cells? How does this relate to residual functions such as cytokine expression?
-What are the causal roles of the metabolic program of CLL T cells in the observed T-cell dysfunction?
-Which epigenetic and transcriptional checkpoints are key in the differentiation towards CLL-induced T-cell dysfunction? And are these events reversible? -Which interaction between the CLL and T cells leads to dysfunction? Is it a contact-dependent interaction, a soluble factor or a combination of both?
DNA methylation
DNA methylation is the addition of a methyl-group (-CH3) to the fifth carbon of cytosines.22 In mammals, this predominantly occurs on a cytosine followed by a guanine in the DNA (e.g., a CpG dinucleotide). Historically, DNA methylation is associated with gene silencing but its function is much more diverse and is highly dependent on the location in the genome. Methylated CpG dinucleotides in and around transcription-factor-binding motifs can prevent binding of methylation-sensitive transcription factors (transcription factors) and thereby silence gene expression, but they can also serve as a binding molecule for transcription activators that recruit DNA demethylating enzymes to allow transcription.22 DNA methylation is actively maintained during cell division by DNA methyltransferase 1 (DNMT1), de novo methylation is performed by DNMT3A/B and loss of methylation happens either passively during cell division or actively by ten-eleven translocation (TET) enzymes.
3D chromatin structure
Chromatin folding occurs on the scale of nucleosomes, chromatin fibers, chromosome domains, compartments and finally on chromosome territories.107 It is a highly complex and multi-level conformation and we are only starting to unravel its role in development and disease. An important feature of this higher-order structure is the interaction between cis-regulatory regions such as enhancers and their target genes, this occurs through formation of chro- matin loops. Enhancer-promoter interactions are mostly constrained within topicalogically associated domains (TADs) which are genomic regions on a sub- megabase scale that show high interaction with each other.107 Chromatin accessibility on the level of nucleosomes can be measured using Assay of Transposase Accessible Chromatin (ATAC) sequencing. Higher-order genome interactions can be studied with methods based on established imaging approaches and chromatin conformation capture (3C) techniques.107
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