Investigating the role of human STING activation in myeloid cells for agonist-induced immune responses

Abstract

The Stimulator of Interferon Genes (STING) intracellular DNA sensing pathway is an important part of the innate immune system which can initiate pro-inflammatory responses. Therapeutic agonism of STING has been shown to induce antigen-specific CD8+ T cells conferring tumour protection in numerous pre-clinical models. However, there are various other outcomes of the pathway, including immune cell death, which may have hindered the efficacy of STING agonist therapy in cancer patients. There is a need for a greater understanding of the regulation of these outcomes and the effects of exogenous STING activation in different cell types.
Myeloid cell STING activation has largely been considered to contribute to pro-inflammatory immune activation and tumour control, but stronger STING signalling can also induce cell death in this cell type and its exact role in STING agonist-mediated anti-tumour responses remains unclear. This project used a novel human (h) STING knock-in mouse model (hSTING flox KI) and compared it to a strain with cre recombinase-mediated myeloid cell specific hSTING deletion (LysMCre/hSTING flox KI) to study the effects of hSTING activation in vivo and decipher the direct contributions of myeloid cell intrinsic STING signalling.
First, these mouse models were characterised ex vivo and compared to WT and STING knock-out murine cells and to human cells. Next, the effects of endogenous and exogenous hSTING activation in myeloid cells on tumour control in vivo were examined. The cyclic dinucleotide-based STING agonist ADU-S100 showed limited potency to activate hSTING but the small molecule diABZI had measurable anti-tumour activity. However, overall modest responses were observed and no clear role for myeloid cell STING was identified.
Recent reports have documented that activated STING functions as a proton pore initiating selected outcomes of the pathway. This included NLRP3 inflammasome activation which proceeds pyroptotic cell death in monocytes. It was also found that a unique transmembrane domain-binding agonist compound (C) 53 can block the engagement of this function by other agonists. Following this, here the effects of diABZI and C53 on myeloid cells in vitro were examined. This revealed that their combination induced unique signalling to either alone.
The role of myeloid cell STING in the induction of antigen-specific CD8+ T-cell responses by hSTING agonism in vivo was also explored using diABZI in immunisation studies. This showed that higher doses of diABZI suppressed CD8+ T-cell proliferation and induced toxicity dependent on monocyte STING. The combination of C53 with high dose diABZI was able to restore the expansion of T cells indicating involvement of a proton channel-mediated function of STING. Blocking IL-1β had similar effect to C53 highlighting a potential role for this downstream effect of NLRP3 inflammasome in monocytes. This thesis is the first extensive exploration of hSTING agonism in vivo and identified a previously uncharacterised detrimental impact of monocyte STING on STING activation driven CD8+ T-cell responses.

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Identifiers

ORCID for Esme Josephine Casson Fowkes: orcid.org/0009-0003-4913-1167

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

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