AbstractInterleukin-10 (IL-10) is a key immuno-regulatory cytokine with potent anti-inflammatory effects. IL-10 contributes to immuno-suppression by inhibiting the presentation of antigen and decreasing the production of pro-inflammatory cytokines from innate cells such as dendritic cells and macrophages which in turn can suppress the activity of adaptive T cells. However, much is still unknown about the precise mechanisms through which this cytokine exerts its effects. This thesis used protein engineering techniques to explore the molecular mechanisms underpinning IL-10’s activities.
IL-10 occurs naturally as a dimeric molecule which binds two receptor subunits: IL-10Rα and IL-10Rß. It has very poor affinity for the IL-10Rß receptor subunit and is unable to bind IL-10Rß in the absence of IL-10Rα (Walter, 2014). Using error-prone yeast display we were able to generate a variant of IL-10 which could directly bind to the IL-10Rß subunit, with affinities in the low micromolar range. This variant was recombinantly expressed in both the monomeric and dimeric conformations. Using these engineered variants in combination with the wild type monomeric and dimeric IL-10, allowed us to examine the contribution of both receptor affinity and stoichiometry to IL-10’s activities.
Single particle imaging of the receptor subunits on the surface of live cells showed that increased affinity for IL-10Rß enhanced heterodimerisation of IL-10Rα and IL-10Rß for both the monomeric and dimeric variants. This increased receptor complex assembly translated into increased STAT1 and STAT3 activation by the high affinity monomer compared to the wild type monomer. The high affinity dimer showed a similar maximal STAT activation compared to the wild type dimer however, it also showed favourable activity at lower concentrations. This resulted in enhanced biological activities by the high affinity dimer compared to the wild type at sub-saturating concentrations e.g., enhanced suppression of LPS-induced IL-6 secretion.
In order to view differences between the variants on a more global level, RNA sequencing was performed on human primary monocytes and CD8 T cells, treated with the monomeric and dimeric wild type and high affinity variants. The high affinity monomer had enhanced regulation of almost all transcripts compared to the wild type monomer however, it failed to reach dimeric expression levels. The wild type dimer showed poorer gene regulation when used at low doses, particularly when looking at suppression of pro-inflammatory cytokines and chemokines in monocytes. The high affinity dimer showed enhanced regulation of subsets of genes at low doses compared to the wild type, suggesting that improved receptor affinity could be used to overcome dose-dependent limitations. These data demonstrate that both stoichiometry and affinity play a large role in IL-10’s activities.
A structure of the complete IL-10/IL-10Rα/IL-10Rß complex is lacking, hindering our understanding of the molecular interactions in this cytokine-receptor complex. We used our high affinity variants to attempt crystallisation of the receptor complex. While a stable complex was successfully purified by gel filtration, no crystals were generated. However, work done here lays the foundation for future attempts using alternate technologies such as cryogenic electron microscopy.
IL-10 has also been shown, under certain conditions, to enhance the tumour targeting ability of CD8 T cells (Naing et al., 2018) however much is still unknown about this process. We used proteomics as well as RNA sequencing to examine the action of IL-10 in human CD8 T cells. This study showed that IL-10 can downregulate exhaustion markers in these cells. In addition, it highlighted downregulation of several enzymes in glycolysis as well as an upregulation of several mitochondrial components, indicating that IL-10 may be regulating metabolic processes in CD8 T cells. While these results are only considered preliminary, they highlight key areas for future work.
|Date of Award||2021|
|Supervisor||Ignacio Moraga Gonzalez (Supervisor) & Doreen Cantrell (Supervisor)|