Characterising transcriptional and epigenetic programming during CD8+ T cell effector and memory differentiation

© 2015 Dr. Michelle Ly Thai NguyenThe hallmarks of CD8+ T cell immunity are the ability to acquire lineage specific effector functions that then promote clearance of foreign pathogens, and the formation of immunological memory upon pathogen clearance. Naïve CD8+ T cells are largely quiescent, however upon antigen stimulation, they undergo proliferation and differentiation resulting in a large pool of effector cells (CTLs) capable of secreting pro-inflammatory cytokines and cytotoxic molecules including Perforin (PRF) and Granzyme A and B (GZMA and GZMB). In contrast, upon subsequent exposure to the same pathogen, memory CTLs rapidly express effector molecules without the need for further proliferation. It is well appreciated that differences in functional capacity between these differentiation states are underscored by coordinated changes in transcriptional profiles. However, the molecular mechanisms that underlie these changes are not fully defined. It has become increasingly clear that modulation of chromatin structure, in particular through post-translational modifications of histone proteins, may play a role in regulating the acquisition and maintenance of lineage specific CTL effector functions, such as the capacity to express high levels of IFNγ, PRF and GZMB. Despite being a signature CTL effector gene, little is known about regulation of Gzma. Therefore, this thesis aimed to dissect the molecular events underpinning the transcriptional activity of Gzma upon CD8+ T cell differentiation. Using an in vitro activation of naïve CD8+ T cells in conjunction with chromatin immunoprecipitation (ChIP), the first chapter demonstrated that the addition of IL-4 enhanced Gzma transcription, and this correlated with STAT6-dependent promotion of a permissive histone signature, and binding of GATA3 and the histone acetyltransferase P300 at the Gzma locus. Interestingly, while GZMA expression by ex vivo isolated effector influenza A virus-specific CTLs was associated with a similar permissive epigenetic signature and GATA3 binding to the Gzma locus, this was independent of STAT6. Given that GATA3 is key for CTL differentiation in response to viral infection, it was hypothesised that GATA3 expression may be regulated independent of IL-4/STAT6 signalling pathway. Indeed, this thesis further demonstrated that in vitro stimulation of naïve CD8+ T cells in the presence of the NOTCH ligand, Delta1, resulted in up-regulation of GZMA expression when compared to CD8+ T cells activated in the absence of NOTCH signals, and that coincided with greater binding of GATA3 to the Gzma locus. However, unlike other effector gene loci, memory CTLs did not maintain permissive histone post-translational modifications at the Gzma locus, even though this was re-established upon recall of memory CTLs following a secondary challenge. Results from this chapter indicate that GZMA expression by virus-specific CTLs is regulated by differentiation-dependent modulation of chromatin composition at the Gzma locus that differs to other signature CTL effector gene loci, and this signature can be influenced greatly by the presence of specific extracellular signals. To further understand how different extrinsic signals are integrated to influence binding of transcription factors and subsequent regulation of the transcriptional program, genome-wide profiling of the transcriptome and GATA3 binding was performed on CTLs cultured in the presence of IL-4, NOTCH or directly isolated after IAV infection. Activation of CD8+ T cells with different signals led to differential GATA3 binding across the genome and this was correlated with distinct transcriptional outcomes. Thus, results obtained from this study suggest that extracellular signals play a crucial role in determining target specificity of GATA3 and that ultimately contributes to the changes in gene expression pattern of CTLs. Taken as a whole, this thesis demonstrated that acquisition of GZMA expression by activated CD8+ T cells is strongly linked to defined chromatin remodelling events at the gene locus and that is, in part, dictated by receipt of specific extrinsic signals from environmental cues that are present during CD8+ T cell differentiation. Importantly, this study found that modulation of chromatin structure is not the generic mechanism regulating the rapid acquisition of effector genes within memory cells, but rather is dependent on the locus being analysed. Finally, this study further reinforced the importance of extracellular signals on CD8+ T cell differentiation; in particular this thesis provides novel insights into the detailed mechanisms of how these extracellular signals may regulate the transcriptional programs and subsequent fate of CTLs upon differentiation. Overall, a comprehensive understanding of regulation of CD8+ T cell mediated immunity would be critical for the designs of immunotherapies and novel vaccine strategies that aim to induce effective and cross-protective CD8+ T cell responses