Periodic Reporting for period 1 - PYRAMID (PYHIN-Regulated Memory T cell protection for Infectious Diseases)
Berichtszeitraum: 2021-09-01 bis 2023-08-31
PYHIN proteins play key roles in innate immunity as viral restriction factors, nucleic acid sensors and transcriptional regulators of cytokines and type I interferons (IFN-I). However, much of the work resolving these roles has been performed in cell lines or macrophages, while little is known about how PYHINs might influence adaptive immunity. Herein, we sought to better understand how PYHINs regulate innate immunity in dendritic cells (DCs), the primary antigen presenting cell, responsible for driving adaptive immunity. The type and magnitude of DCs response to noxious stimuli has a profound effect on the ensuing T cell response, both in terms of its quality and longevity. An inability to drive robust T cell responses is a limitation of many licensed vaccines, including current seasonal influenza vaccines, and underpins the attenuated protection they confer against divergent or newly emerging strains or variants. Thus, a better understanding as to how these responses are regulated will aide the rational design of next generation vaccines capable of inducing robust T cell responses. The overall objective of this project is to understand how PYHIN proteins contribute to innate immune responses in DCs, via three distinct work-packages:
Characterise PYHIN expression in DC cell subsets before and after stimulation with known pathogen-associated molecular patterns (PAMPs).
Determine whether altering PYHIN expression influences innate cytokine and IFN-I secretion from DCs following exposure to viral PAMPs.
Assess the contribution of PYHINs to T cell responses in vivo.
Major Conclusions:
Type 1 and type 2 DCs exhibit different patterns of expression of PYHINs e.g Ifi205 almost exclusively expressed in DC1 at both the mRNA and protein level.
Deletion of the entire murine PYHIN locus (or Aim2 alone) results in enhanced pro-inflammatory and IFN-I responses in response to transfected DNA, but not other stimuli including RNA or toll-like receptor (TLR) agonists. This enhanced responsiveness includes increased activation of several intracellular signalling pathways.
The enhanced IFN-I response seen in Aim2-/- and ALR-/- DCs leads to enhanced antigen processing and up-regulation of co-stimulatory molecule expression.
In vivo, treatment of tumour-bearing ALR-/- mice with a proprietary cationic polymer results in tumour eradication.
Characterise PYHIN expression in DC cell subsets before and after stimulation with known pathogen-associated molecular patterns (PAMPs).
Determine whether altering PYHIN expression influences innate cytokine and IFN-I secretion from DCs following exposure to viral PAMPs.
Assess the contribution of PYHINs to T cell responses in vivo.
Major Conclusions:
Type 1 and type 2 DCs exhibit different patterns of expression of PYHINs e.g Ifi205 almost exclusively expressed in DC1 at both the mRNA and protein level.
Deletion of the entire murine PYHIN locus (or Aim2 alone) results in enhanced pro-inflammatory and IFN-I responses in response to transfected DNA, but not other stimuli including RNA or toll-like receptor (TLR) agonists. This enhanced responsiveness includes increased activation of several intracellular signalling pathways.
The enhanced IFN-I response seen in Aim2-/- and ALR-/- DCs leads to enhanced antigen processing and up-regulation of co-stimulatory molecule expression.
In vivo, treatment of tumour-bearing ALR-/- mice with a proprietary cationic polymer results in tumour eradication.
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.
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.
Despite previous reports identifying PYHIN proteins as regulators of IFN-I gene transcription, this project has surprisingly identified them as negative regulators of the DNA-induced IFN-I cascade in DCs. AIM2 in particular appears to play a crucial role in this regard as deletion of Aim2 alone phenocopied the ALR-/- cells which lack all 13 PYHIN members. While previous studies have reported inhibition of the cGAS-STING pathway due to activation of the AIM2-inflammasome, our studies were performed in the absence of a priming signal and thus inflammasome activation, confirmed by immunoblotting and ELISA. Ongoing work is focused on how inhibition of cGAS, but not STING, impairs the IFN-I response to DNA in Aim2-/- and ALR-/- cells, results which will be included in our manuscript.
Based on our finding that PYHIN proteins negatively regulate DNA-induced DC maturation, we plan to perform extensive analyses how this modulates T cell subset differentiation and survival in a number of contexts. Furthermore, we plan to follow-up on our remarkable in vivo finding to determine whether inhibition of PYHIN-mediated immune regulation may be an adjunct strategy to immunotherapy to boost anti-tumour (and anti-viral) T cell responses. Thus this project has yielded results which can advance our understanding as to how DCs sense and respond to infection or cellular damage to drive T cell responses. Hence targeting the PYHINs themselves (or elements of the pathways they regulate downstream) for therapeutic purposes may prove beneficial to boost anti-viral or anti-tumour immunity or to immune-mediated pathologies e.g following DNA-damaging immunotherapy.
Based on our finding that PYHIN proteins negatively regulate DNA-induced DC maturation, we plan to perform extensive analyses how this modulates T cell subset differentiation and survival in a number of contexts. Furthermore, we plan to follow-up on our remarkable in vivo finding to determine whether inhibition of PYHIN-mediated immune regulation may be an adjunct strategy to immunotherapy to boost anti-tumour (and anti-viral) T cell responses. Thus this project has yielded results which can advance our understanding as to how DCs sense and respond to infection or cellular damage to drive T cell responses. Hence targeting the PYHINs themselves (or elements of the pathways they regulate downstream) for therapeutic purposes may prove beneficial to boost anti-viral or anti-tumour immunity or to immune-mediated pathologies e.g following DNA-damaging immunotherapy.