Investigation of the role of cGAMP in differentiation of T lymphocytes
Author
Yıldız, Begüm
Advisor
Gürsel, İhsan
Date
2016-10Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
STING is the pivotal mediator for the recognition of host and pathogenic cytosolic
dsDNA as well as cyclic di-nucleotides metabolites from microbes. STING can either
recognize DNA itself or sense the presence of cGAMP, which is converted from ATP
and GTP upon DNA binding to cGAS enzyme. Not only strategy against intracellular
pathogens makes STING an ideal target, but also the recognition of DNA from host cells
has a significant role in tumor immunity. Previous studies demonstrated that DNA
released from cancerous cells are internalized by innate immune cells such as
macrophages and dendritic cells in tumor microenvironment and trigger the production of
IFN-β and other pro-inflammatory cytokines including IL-6, TNF-α, and IL-12 through
STING triggered signaling pathway. These cytokines then enhance cytotoxic activity of
CD8+ T cells by further increasing IFNγ production. Since enhanced T cell immunity is
the hallmark of vaccine adjuvants, cyclic di-nucleotides such as cGAMP become an
important and effective vaccine adjuvants against intracellular pathogens and malignant
cells. Although STING activating cyclic di-nucleotides are envisioned as novel and potent vaccine adjuvants, more thorough research is needed to unearth the mechanism of
action of STING on different immune cells. Therefore, it will pave the way for the
initiation of successful human trials. The important criteria while developing vaccine
adjuvant are the magnitude, and the quality of an immune response and its toxic side
effects. To identify these, members of the both innate and adaptive immune system
should be taken into account. However, previous studies merely focus on the function
and effect of cGAMP in innate immune cells such as macrophages, monocytes and
dendritic cells. However, to date there is no explicit study investigating the effect of
STING signaling cascade on T-cells. In the light of these findings, we aimed to
investigate the direct effect and function of cGAMP on T lymphocytes. Since there were
not any preliminary data, we firstly stimulated Pan T cells with cGAMP alone or together
with various TLR ligands and then, checked the cytokine profiles and the viability of
cells. Surprisingly, 2.5µg/ml dose of cGAMP had a toxic effect on T cell but not on bone
marrow derived dendritic cells and macrophages. While cGAMP triggered cell death,
interestingly IL-17 secretion from both CD4+ and CD8+ T cells was dramatically
increased. Beside, cGAMP stimulation drastically increased CD4+
/CD8+ T cells ratio of
Pan T cells population. Next, we sought to identify the source of IL-17. The IL17
inductive capacity of cGAMP was investigated on purified CD4+ T cells from mice.
Unexpectedly, data revealed that cGAMP elicited apoptosis of CD4+ T cells. Moreover,
there was no significant induction of IL-17 secretion. Next, we aimed to find a condition
that will reduce the toxic effect of cGAMP, while maintaining IL-17 secretion. When Pan
T cells were stimulated with cGAMP and R848 (a TLR7 ligand), the toxic action of
cGAMP decreased while IL-17 secretion was enhanced. Lastly, the potency of T cells
stimulated with cGAMP was investigated. According to our results, macrophages were
activated in the presence of conditioned medium obtained from T cells stimulated with
cGAMP. When taken together our findings point out that STING dependent direct
activation of T-cells via cGAMP and its subsequent effect on macrophages might be
utilized as an immunotherapeutic approach where IL17 induction is important and could be harnessed as vaccine adjuvants against mucosal infections or against cancer.