We first optimised two independent protocols for differentiating bone marrow progenitor cells into DCs with GM-CSF or GM-CSF plus FLT3L, the latter yielding DCs phenotypically and functionally similar to those which cross present antigen to drive CD8+ T cell responses. Both DC types were differentiated from the bone marrow of ALR-/-, Ifi204-/-, Ifi207-/- and Aim2-/- mice and responses compared to DCs from wild-type (WT) controls following stimulation. Using this approach we made the surprising discovery that in contrast to their positive role in DNA sensing in other cells, PYHIN proteins serve as powerful negative regulators of the innate immune response to DNA in DCs. Upon stimulation of DCs with transfected DNA, we consistently observed increased production of pro-inflammatory cytokines and IFN-I from ALR-/- DCs compared to WT cells. We also observed increased activation of key intracellular signalling molecules including STING, TBK1, IRF3 and NFκB in ALR-/- cells following stimulation with DNA, but not other stimuli. Furthermore, we determined that the enhanced IFN-I produced by ALR-/- DCs signalled in an autocrine manner, resulting in increased expression of co-stimulatory markers such as CD86 on the surface of ALR-/- DCs and an enhanced capacity to process antigen as determined by flow cytometry. While these phenotypes were not apparent in Ifi204-/- and Ifi207-/- DCs, we hypothesised that AIM2 may be implicated as it is known to sense DNA. Through collaboration with Prof Jose Bengoechea at Queens University Belfast, we established an Aim2-/- mouse colony at Trinity College Dublin (TCD) and successfully reproduced the phenotype observed previously in ALR-/- DCs, suggesting that AIM2 may serve as a competitive inhibitor to cGAS in the context of DNA sensing. Inhibition of cGAS and STING did indeed reduce the production of NFκB-dependent cytokines such as IL-6 and TNFα by Aim2-/- and ALR-/-DCs, however, inhibition of STING only partially impaired the IFN-I response and failed to prevent phosphorylation of TBK1 and IRF3, both downstream of STING in the cGAS-STING DNA sensing pathway.
To test the enhanced responsiveness of ALR-/- cells to DNA in vivo, we collaborated with the Adjuvant Research Group at TCD and performed two independent therapeutic cancer studies whereby WT and ALR-/- mice were implanted with tumour cells prior to treatment with proprietary polymers known to induce IFN-I-dependent responses via endogenous DNA release. Remarkably, while polymer treatment alone induced modest but significant protection in WT mice, tumours were completely eradicated in ALR-/- mice.
The results of this project have been presented at as oral presentations at the Host-Pathogen Communication Conference 2021 and Irish Society of Immunology annual meeting 2022 and as posters at the British Society for Immunology Congress (BSI) in 2021 and 2022. Abstracts have been accepted for BSI and for a symposium on Innate Immunity in Viral Infection at Leiden University both in December 2023. A manuscript on this work is in preparation (expected submission January 2024). These results formed the basis for a successful application for funding through The Royal Society University Research Fellowship Scheme, thus enabling me to establish my own independent research group at TCD.
