Molecular Regulation of Memory T Cell Differentiation and Function

Scamardella, Eloise (2020). Molecular Regulation of Memory T Cell Differentiation and Function. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00011edc

Abstract

Memory T cells are largely heterogeneous. The molecular mechanisms at the basis of their effector functions are still under investigation. FACS-sorted memory T cell subsets exposed to TCR activation indicated that IRF8 is upregulated rapidly in all T cells, but its expression is rapidly lost, hence suggesting a role during the early stages of effector T cell programming. The aim of the first part of the work was to dissect the biological role of IRF8 in CD8+ T cells. I transferred Irf8-/- or Irf8+/+ CD8+ T cells in recipient WT mice, then infected those mice with LCMV. My results showed that Irf8 deficiency led to a faster transition towards memory phenotype after the acute phase. IRF8 silencing in human CD8+ T cells, followed by transcriptomic analysis, revealed a down regulation of effector related genes, accompanied by the upregulation of key memory TFs (FOXO1, MYB, TCF7). Accordingly, overexpressing IRF8 in primary human CD8+ T cells resulted in FOXO1 repression. ChIP sequencing, performed in IRF8-overexpressing Jurkat cells, identified a peak of IRF8 binding on FOXO1 promoter region. Taken together, I conclude that IRF8 restrains FOXO1 expression by binding directly to the chromatin.

Adoptive T cell transfer immunotherapy benefits from early differentiated memory T cells capable to persist in the long term and to generate anti-tumour effectors. Cellular differentiation, metabolism and survival can be regulated by reactive oxygen species, but the mechanism is still poorly understood. I found that cellular antioxidant capacity is higher in human CD8+ T naïve (T_N) cells compared to more differentiated memory T cell subsets. I expanded human CD8+ T_N cells in vitro in the presence of ROS scavenger N-acetyl-cysteine (NAC) and found that limiting global ROS metabolism during T_N cell activation hindered effector differentiation and enabled the generation of stem-like memory T cell precursors. I thus define a novel mechanism to generate potent T cell memory based on limiting ROS metabolism and propose to use NAC as suitable strategy to expand T cells for ACT immunotherapy.