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Comparative Molecular, Innate, and Adaptive Impacts of Chemically Diverse STING Agonists 5
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Nobuyo Mizuno1, Dylan Boehm1, Kevin Jimenez-Perez1, Jinu Abraham1, Laura Springgay1, Ian Rose1, Victor 12
R. DeFilippis1*
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1Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, United States 18
of America. 19
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*To whom correspondence should be addressed: defilipp@ohsu.edu21
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version;Title_Page.docx
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.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.21.639458doi: bioRxiv preprint
ABSTRACT
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Pharmacologic activation of the innate immune response is being actively being pursued for numerous clinical
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purposes including enhancement of vaccine potency and potentiation of anti-cancer immunotherapy. Pattern
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recognition receptors (PRRs) represent especially useful targets for these efforts as their engagement by
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agonists can trigger signaling pathways that associate with phenotypes desirable for specific immune outcomes.
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Stimulator of interferon genes (STING) is an ER-resident PRR reactive to cyclic dinucleotides such as those
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synthesized endogenously in response to cytosolic dsDNA. STING activation leads to transient generation of
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type I interferon (IFN-I) and proinflammatory responses that augment immunologically relevant effects including
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antiviral responses, antigen presentation, immune cell trafficking, and immunogenic cell death. In recent years
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engineered cyclic dinucleotides and small molecules have been discovered that induce STING and safely confer
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clinically useful outcomes in animal models such as adjuvanticity of anti-microbial vaccines and tumor clearance.
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Unfortunately, clinical trials examining the efficacy of STING agonists have thus far failed to satisfactorily
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recapitulate these positive outcomes and this has prevented their translational advancement. A likely relevant
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yet perplexingly under investigated aspect of pharmacologic STING activation is the diversity of molecular and
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immune responses that associate with chemical properties of the agonist. Based on this, a comparative survey
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of these was undertaken using unrelated STING-activating molecules to characterize the molecular, innate,
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cellular, and immune outcomes they elicit. This was done to inform and direct future studies aimed at designing
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and selecting agonists appropriate for desired clinical goals. This revealed demonstrable differences between
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the agonists in potency, transcriptomes, cytokine secretion profiles, immune cell trafficking, and antigen-directed
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humoral and cell mediated immune responses. As such, this work illustrates that phenotypes deriving from
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activation of a protein target can be linked to chemical properties of the engaging agonist and thus heightened
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scrutiny is necessary when selecting molecules to generate specific in vivo effects.
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Keywords: STING, adjuvant, cytokines, vaccines, interferon.
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Click here to access/download;Abstract;Abstract.docx
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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ABSTRACT
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Pharmacologic activation of the innate immune response is being actively being pursued for numerous clinical
24
purposes including enhancement of vaccine potency and potentiation of anti-cancer immunotherapy. Pattern
25
recognition receptors (PRRs) represent especially useful targets for these efforts as their engagement by
26
agonists can trigger signaling pathways that associate with phenotypes desirable for specific immune outcomes.
27
Stimulator of interferon genes (STING) is an ER-resident PRR reactive to cyclic dinucleotides such as those
28
synthesized endogenously in response to cytosolic dsDNA. STING activation leads to transient generation of
29
type I interferon (IFN-I) and proinflammatory responses that augment immunologically relevant effects including
30
antiviral responses, antigen presentation, immune cell trafficking, and immunogenic cell death. In recent years
31
engineered cyclic dinucleotides and small molecules have been discovered that induce STING and safely confer
32
clinically useful outcomes in animal models such as adjuvanticity of anti-microbial vaccines and tumor clearance.
33
Unfortunately, clinical trials examining the efficacy of STING agonists have thus far failed to satisfactorily
34
recapitulate these positive outcomes and this has prevented their translational advancement. A likely relevant
35
yet perplexingly under investigated aspect of pharmacologic STING activation is the diversity of molecular and
36
immune responses that associate with chemical properties of the agonist. Based on this, a comparative survey
37
of these was undertaken using unrelated STING-activating molecules to characterize the molecular, innate,
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cellular, and immune outcomes they elicit. This was done to inform and direct future studies aimed at designing
39
and selecting agonists appropriate for desired clinical goals. This revealed demonstrable differences between
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the agonists in potency, transcriptomes, cytokine secretion profiles, immune cell trafficking, and antigen-directed
41
humoral and cell mediated immune responses. As such, this work illustrates that phenotypes deriving from
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activation of a protein target can be linked to chemical properties of the engaging agonist and thus heightened
43
scrutiny is necessary when selecting molecules to generate specific in vivo effects.
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Keywords: STING, adjuvant, cytokines, vaccines, interferon.
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.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.21.639458doi: bioRxiv preprint
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INTRODUCTION
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Subunit vaccine antigens when used alone do not typically exhibit levels of immunogenicity that are sufficient for
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the establishment of protective immunity. As such, they require coadministration of factors such as adjuvants to
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stimulate the innate immune system in a manner resembling that which occurs during exposure to molecular
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signals of proliferative microbial danger [1]. Unfortunately, only a small number of adjuvants are approved for
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clinical use and effective protection requires pathogen-specific immune polarization such that one adjuvant is
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unlikely to achieve all objectives [2]. Moreover, the precise mechanisms of action of clinical and experimental
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vaccine adjuvants are often poorly characterized [3]. The need is therefore great for pursuit and characterization
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of new adjuvant classes, especially due to the likelihood of pathogen emergence and the number of established
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infections for which no or suboptimal vaccines exist. A promising investigative approach to adjuvant discovery is
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identification of pharmacologic activators of pattern recognition receptors (PRRs) [4]. Engagement of PRRs by
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pathogen- or danger-associated molecular patterns initiate intracellular signaling processes that culminate in
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innate immune activation. This includes proinflammatory cytokine secretion and activation of antigen presenting
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cells (APC) that facilitate and direct antigen-directed adaptive immune responses.
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Stimulator of interferon genes (STING) is an ER-resident PRR reactive to cyclic purine dinucleotides (CDNs)
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synthesized by bacterial and eukaryotic nucleotidyl cyclases. Most prominently, cyclic GMP-AMP (cGAMP)
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synthase (cGAS) is a vertebrate enzyme activated following contact with cytosolic DNA to synthesize 2’3’ cGAMP
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from ATP and GTP. Activated STING leads to phosphorylation of TANK binding kinase 1 (TBK1) which in turn
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phosphorylates transcription factors such as interferon regulatory factor 3 (IRF3) and nuclear factor B (NF-B).
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These leads to expression of type I interferons (IFN-I) as well as other pro-inflammatory cytokines and
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costimulatory factors. In addition, STING is expressed across a wide range of cell types and induces
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transcription-independent processes such as autophagy, inflammasome induction, and lysosomal biogenesis
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often in ways that are cell- and agonist-specific [5–8] . These collectively contribute to the innate immune
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phenotypes conferred by activated STING and facilitate the establishment of adaptive immune responses
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through localized recruitment and maturation of APCs, antigen loading and processing, and antigen presentation
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in the draining lymph node (dLN). STING is therefore an increasingly attractive cellular target for development
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of new adjuvants and its stimulation as such has shown to enhance efficacy of vaccines against diverse pathogen
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types.
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Multiple CDN classes can activate STING and enhance vaccine-mediated immune responses in animal models.
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Furthermore, synthetic small molecules have also been identified that induce STING and adjuvant functions
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have been described for many of these. Numerous efforts are currently underway to thus discover and
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characterize novel STING-activating molecules for similar use. However, liabilities associated with certain
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chemical classes can affect their in vivo suitability, potency, and performance. For instance, CDNs are
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susceptible to degradation by phosphodiesterases and also cross cell membranes poorly due to their size and
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hydrophilicity. More importantly, the profile of STING-mediated molecular processes is influenced by the
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chemical structure of the ligand. This may be due to a number of causes including engagement of off-target
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proteins, pharmacokinetics, region of binding, or molecule-specific conformational changes in STING that affect
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its activity. As such, it is likely insufficient to simply target STING using any activating ligand since the consequent
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immune phenotypes could vary substantially and confer outcomes inconsistent with a vaccine’s purpose.
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This study aims to undertake in vitro and in vivo characterization of three well described but chemically dissimilar
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STING agonists. Our purpose is to perform direct comparisons of molecular, innate, and adaptive immune
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responses that they induce to identify effects that are both shared and divergent between them. We hope these
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will inform future studies that pursue STING inducers for specifically defined clinical outcomes. This work also
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highlights multi-level phenotype patterns correlating with STING activation that were unexpected in our models.
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RESULTS
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Dose-dependent activity of STING agonists in vitro
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To compare molecular, innate, and adaptive immune phenotypes elicited by chemically dissimilar STING ligands
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we used cyclic di-AMP analog ML-RR-S2 CDA (CDA) [9], the amidobenzimidazole diABZI [10], and 5,6-
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dimethylxantheonone-4-acetic acid (DMXAA) [9,11,12]. We first characterized the potency, patterns of innate
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activation, and cytotoxicity to allow identification of suitable doses for subsequent in vivo use. For this we used
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RAW264.7 and J774 macrophage-like murine cell lines engineered to express luciferase in response to IFN-I-
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dependent signaling [13,14]. These were exposed in duplicate to a range of concentrations of the molecules and
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luciferase signal measured after 24 h. CDA was transfected since it is not easily cell permeable due to its size
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and hydrophilicity. In parallel, cytotoxicity was also measured using the ATP-based Cell Titer Glo reagent. As
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shown in Figures 1A and 1B, differences were observed between the cell types primarily at the highest
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concentrations of each molecule with RAW264.7 displaying overall lower viability than J774 cells. Moreover,
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J774 cells also showed greater sensitivity to the compounds as indicated by higher overall fold changes in
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luciferase induction for all agonists. Additionally, CDA elicited the highest peak induction levels in both cell types.
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J774 cells co-express secreted alkaline phosphatase (SEAP) in response to NF-kB activation and we therefore
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also examined this response. As shown in Figure 1C, detectable SEAP was observed at the highest dose of
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DMXAA and at multiple doses of CDA but was not detected at any diABZI dose. Importantly, these results
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indicate that levels of cytotoxicity at doses eliciting peak innate induction in both cell types were not unsuitably
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low indicating that these were appropriate for use in additional studies. Moreover, NF-kB activation was only
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significantly detected in response to CDA, highlighting a key difference in phenotypic response to the agonists.
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Activation of STING is also known to stimulate the NLRP3 inflammasome, which can greatly affect the
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proinflammatory tissue environment through secretion of IL-1 and IL-18 [5,15]. However, the degrees to which
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this is induced by different agonist chemical classes has not been examined. We therefore used the J774
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monocyte cell line as has been previously described for NLRP3 inflammasome studies to assess the response
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to these agonists [16,17]. In duplicate cells were primed for 5 h with LPS and exposed overnight to a dosing
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range of diABZI, CDA, or DMXAA. Secretion of IL-1 was then measured using bead-based Luminex assay. As
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shown in Figure 1D, all three agonists stimulated secretion of IL-1 although with divergent induction patterns
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that were maximal near 0.032 µM. However, while diABZI secretion declined linearly with higher agonist doses,
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DMXAA and CDA generated dissimilarly sigmoidal decreases with higher dosage. Based on these results we
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conclude that the agonists collectively stimulate qualitatively different patterns of intracellular innate induction as
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indicated by degree of dose-dependence, cytokine (IFN-I and IL-1) secretion, and NF-B activation.
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STING agonist-mediated dendritic cell maturation
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Adjuvant-enhanced adaptive immune processes require the local activation of dendritic cells (DC) to enable
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antigen uptake and presentation on MHC-II to CD4+ helper cells in the draining lymph node (dLN) [18]. We
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therefore compared the ability of STING ligands to induce the expression of MHC-II and costimulatory proteins
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CD86, CD80, and CD40. For this we used nontoxic concentrations of each molecule near those that displayed
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the highest reporter signal in in vitro assays (Figure 1). Control stimuli included LPS and Alum as respective
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inducers of DC activation and subunit vaccine response enhancement. Immature bone marrow derived DC
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(BMDC) from C57Bl/6 mice were treated overnight and flow cytometry used to measure surface expression of
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the markers. As shown in Figure 2, all STING ligands as well as LPS led to significant upregulation of the four
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markers relative to DMSO-treated cells whereas no such induction was observed for Alum. In addition, DMXAA
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appears capable of eliciting significantly higher levels of expression of these proteins than the other STING
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ligands and (with the exception of MHC-II) LPS. As such, these STING agonists are highly but differentially
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effective at inducing maturation of DC in an ex vivo system, a process necessary for establishing adaptive
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immune responses.
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In vivo cytokine induction by STING agonists
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Adjuvant mechanisms associate with stimulation of innate processes immediately following in vivo
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administration. While administered locally, this often manifests as secretion of proinflammatory cytokines that
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are detectable in peripheral blood [19,20]. We therefore compared this indicator of systemic innate immune
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induction by the three molecules using secretion of peripheral cytokines as a readout. Using doses that are
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consistent with what was found to induce near maximal IFN-I levels, we injected each compound intramuscularly
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(IM) into C57Bl/6 mice to simulate a typical vaccination route using PBS as a negative control injection. Mice
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were euthanized at 24 h and peripheral blood collected. Levels of cyto/chemokines were then measured in serum
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using Luminex multiplex bead-based assay. As shown in Figure 3, three different cytokine induction patterns
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were detected for the stimuli. Secretion of cytokines such as CCL7 (MCP-3) and IL-1 was significantly and
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similarly induced by all three treatments relative to control mice. Cytokines such as CXCL10 and IL-18 were
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significantly stimulated by CDA and DMXAA but not diABZI. Finally, DMXAA alone induced significant secretion
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of CCL2, CCL3, CCL4, CCL5, CCL11, CXCL1, CXCL2, IL-6, IL-10, IL-12p70, IL-17A, IL-22, IL-27, and TNF.
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These results suggest that while some innate pathways such as those leading to secretion of CCL7 and IL-
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1are stimulated comparably in response to all stimuli, others can be differentially activated in an agonist-
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specific manner. Moreover, the response to DMXAA appears unusual among the agonists in that it is uniquely
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capable of inducing secretion of a subset of cytokines. This is underscored by the fact that no secretion of CCL3,
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IL-10, IL-17A, IL-22, and IL-27 above the limit of detection was observed in response to other agonists for yet
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they were significantly stimulated by DMXAA.
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In vivo transcriptional impact of STING agonist administration
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The rapid innate processes conventionally induced by adjuvants are most highly localized to the site of
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administration and dLN where adaptive immune processes commence. We next examined this at the
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transcriptional level by injecting mice with diABZI, CDA, DMXAA, or PBS as described above. Total RNA was
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isolated from dLN harvested at 5 h, 24 h, or 72 h and semi-quantitative RT-PCR (qPCR) used to measure
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adjuvant-dependent upregulation of mRNAs encoding conventional IFN-stimulated and pro-inflammatory
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proteins. As shown in Figure 4A, transcriptional induction peaked for nearly all genes at 5 h and declined in later
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time points although at different rates between agonists. Importantly, levels of all examined mRNAs displayed
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significant differences between at least two stimuli at multiple time points with the most differences observed
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between DMXAA and CDA and the fewest between DMXAA and diABZI. Moreover, DMXAA stimulation led to
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the highest mean induction of all genes.
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Based on these results we decided to obtain a global picture of changes occurring in whole dLN transcriptomes
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in response to IM injection of the three agonists. For this we undertook a high content mRNA hybridization array
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experiment encompassing >21,000 mouse transcripts. Given the degree of temporal diminishment in
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transcriptional induction (Figure 4A), we examined only 5 and 24 h time points. The number of mRNAs showing
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significant up- or downregulation in dLN harvested following agonist injection were determined by comparing
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absolute signal levels of mRNA-specific probe binding between STING agonist and PBS injected mice to obtain
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transcript fold change (Supplemental Table 1). As indicated in Figure 4B, DMXAA triggers a stronger
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transcriptional response relative to the other molecules as indicated by the number of significantly induced and
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repressed mRNAs at both time points. This is also consistent with what is observed by qPCR of targeted genes
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(Figure 4A). Sample clusters obtained using principal component analysis (PCA) further indicated that the
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transcriptomes generated in response to DMXAA at both time points were substantially more divergent than for
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the other two stimuli (Figure 4C). Consistent with this, at 5 h, stimulation with CDA and diABZI led to upregulation
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of fewer shared transcripts (779) than were uniquely induced by DMXAA (1642). However, at 24 h diABZI
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activated considerably more transcripts than did CDA, a larger portion of which were uniquely shared with
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DMXAA. Over both time points CDA displayed the response of the smallest magnitude and the fewest uniquely
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induced genes (Figure 4D).
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We next used pathway analysis to complement quantitative methods and obtain a context-oriented
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understanding of the biological activities potentially affected by STING agonist administration (Supplemental
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Table 2). We observed within these results pathways similarly activated by all three agonists as shown in Figure
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4E. Intriguingly, pathways were also detected that were only induced by DMXAA at both 5 h and 24 h (Figure
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4F). This includes RAN (Ras-related nuclear protein) signaling and Folate transformation pathways, which were
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predicted to be uniquely induced after both 5 h and 24 h DMXAA treatment. RAN signaling is known to confer
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inhibitory effects on T cell function largely due to impaired nuclear accumulation of AP-1 transcription factors (c-
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Jun, c-Fos) [21]. Moreover, folate is known to be important for proliferation of CD8+ T cells and may impact
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CD4+/CD8+ T cell ratio [22]. These results demonstrate that chemically dissimilar agonists are capable of
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activating transcription that functionally associates with STING stimulation as well as unique pathways linked
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with immunologically impactful processes. As such, agonists may confer differential effects on establishment of
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T cell mediated immune responses.
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Adjuvant-associated changes in cell activation and recruitment to dLN
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Innate immune activating adjuvant administration leads to kinetic, activation, and migratory changes in cellular
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populations at the site of injection and in the dLN [23–26]. This is especially true of APCs such as DC that are
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essential for initiating antigen-specific T and B cell activation and subsequent differentiation. Importantly, STING
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activation has been shown to inhibit growth and even induce death of T, B, and monocytic cells [27–30]. How
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this occurs and its impacts in the context of transient pharmacologic STING activation during vaccination is not
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known. Given the strong transcriptional response observed in dLN, we used flow cytometry to examine changes
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in cell populations here 24 h after intramuscular administration of the antigen ovalbumin (OVA) in the presence
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or absence of CDA, diABZI, and DMXAA using duplicate experiments (Supplemental Figure 1). Alum was used
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as a STING- and IRF3-independent control treatment. As shown in Figure 5A, total dLN cell numbers were not
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significantly altered in response to administration of any stimulus. However, CDA and diABZI led to significant
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increases and DMXAA significant decreases in B (CD3-, CD19+) cell composition of dLN while Alum had no
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effect (Figure 5B). DMXAA also led to a significant decrease in the percentage of T (CD3+, CD19-) cells in the
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dLN while no other stimulus had an effect (Figure 5C). Interestingly, when we examined DC populations, DMXAA
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led to significant increases in frequencies of cDC1 (CD3/B220/CD11b-, CD11c/CD8+), cDC2 (CD3/B220-,
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CD11c/CD11b+), and pDC (CD3/CD11b/CD8-, B220/CD11c+) whereas no such changes were observed for the other
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agonists as shown in Figure 5C-E. In addition, DMXAA was also uniquely associated with increases in dLN-
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localized neutrophils (CD3/B220/F4/80/CD11c-, CD11b+/Ly6G+; Figure 5D) and macrophages
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(CD3/B220/CD11c-, F4/80/CD11b+; Figure 5D). To understand the mechanistic basis of these unusual DMXAA-
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mediated observations we asked whether IFN-I responses are required. For this we used mice lacking the IFN
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receptor (IFNAR-/-). WT and IFNAR-/- mice were treated intramuscularly with OVA alone or admixed with DMXAA.
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As shown in Figure 5C, functional IFN-I signaling was required for increased DMXAA-dependent dLN trafficking
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of cDC1, cDC2, and pDC but not neutrophils or macrophages. Moreover, IFN-I signaling was also required for
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DMXAA-dependent decreases in dLN levels of B and T cells. Based on this we conclude that DMXAA induces
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changes in dLN cell population that are both dependent on and independent of IFN-I signaling. Given the unique
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transcriptomic and cytokine profiles observed in response to DMXAA it is probable that other factors are also
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highly relevant and this will require additional investigation.
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STING ligand-associated enhancement of humoral responses
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Given the obvious innate induction stimulated by STING ligands we next compared the extent to which they
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augment elicitation of antibody responses when co-administered with protein antigen. For this we used a
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prime/boost strategy via intramuscular injection of OVA. As shown in Figure 6A, all ligands led to titers of OVA-
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reactive total IgG that were significantly higher than observed when OVA was injected in the absence of adjuvant.
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Additionally, these levels did not significantly differ from those observed when Alum was used. We next examined
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levels of IgG1 and IgG2c subtypes as an indicator of class switching and T helper (Th) polarization. As shown
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in Figures 6B and 6C, while IgG1 levels for each adjuvant followed the same pattern as total IgG, levels of
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IgG2c were significantly higher for the STING ligands relative to Alum. This is consistent with what is known
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about strong Th2 biased immune phenotypes observed for Alum [31] and Th1 biased responses observed for
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CDA [32]. These results additionally indicate that the small molecule STING ligands diABZI and DMXAA
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generate T helper polarization that more closely resembles CDA than Alum. Surprisingly, levels of IgG1 but not
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IgG2c induced by diABZI were significantly lower than those induced by the other STING ligands. This may
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indicate differential induction of immune processes by the molecule that are mediated by STING or other
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unknown cellular targets. Regulation of antibody production and affinity maturation is mediated in large part
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through the function of T follicular helper (TFH) cells whose activity can be enhanced by the use of adjuvants [33].
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We therefore asked whether adjuvant-specific differences are evident in TFH activation measured following ex
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vivo stimulation of splenocytes with OVA-inclusive peptides. As shown in Figure 6C, DMXAA but not the other
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agonists was able to significantly increase OVA-specific TFH activity following vaccination (Supplemental Figure
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2). Whether this leads to measurable differences in generation of antibody avidity will require additional studies.
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STING ligand-associated enhancement of cell mediated responses
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Previous vaccination models have shown enhanced antigen-directed T cell activation when STING agonists are
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used as adjuvants [32,34,35]. We therefore used IFN ELISPOT to examine whether any differences are
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observed between the ligands with respect to OVA-directed T cell activation. In duplicate experiments animals
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were vaccinated as described above and harvested seven days after boost. Splenocytes were then stimulated
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ex vivo using either an OVA-inclusive peptide pool or the MHC class I immunodominant OVA peptide SIINFEKL.
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As shown in Figure 7A, stimulation with the peptide pool led to significant expression of IFN in splenocytes
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harvested from animals adjuvanted with CDA or DMXAA relative to naïve animals or those vaccinated with OVA
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+ PBS. Moreover, CDA adjuvant also led to significant IFN expression relative to all other adjuvants following
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stimulation with SIINFEKL. Expectedly Alum was also unable to elicit any observed IFN expression. We next
264
used flow cytometry of splenocytes stimulated with OVA peptide pools to distinguish polyfunctional activation in
265
CD4+ and CD8+ T cells as indicated by expression levels of IFN and TNF measured following intracellular
266
cytokine staining (ICS) (Supplemental Figure 3). As shown in Figure 7B, significant expression of TNF and
267
IFN was observed in CD4+ and CD8+ T cells only following vaccination with CDA. These results suggest that
268
chemical structure of STING ligand associates with differential enhancement of antigen-directed T cell activation.
269
270
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12
DISCUSSION
272
Transient pharmacological activation of STING has been shown in animal models to confer desirable immune-
273
mediated outcomes for numerous, seemingly unrelated clinical conditions. This includes enhancement of
274
protective responses conferred by vaccines against viral [35–38], bacterial [39–41], and protozoal [34,42]
275
pathogens. STING agonists have also been shown to impair acute replication of diverse virus types in vivo when
276
administered before challenge [43–47]. Moreover, a wide variety of STING agonist platforms have shown
277
remarkable efficacy in mediating T (reviewed in [48]) and NK [49,50] cell-mediated clearance of transplanted
278
tumors including the generation of abscopal responses [9,51,52]. Intriguingly, pharmacologic STING activation
279
has also been shown to alleviate pain (nociception) via IFN-mediated effects on sensory neurons [53] and to
280
attenuate experimental autoimmune encephalitis [54,55]. As such, STING-dependent phenotypes associate with
281
cellular and immunological processes that can be harnessed for diverse clinical benefits and thus pursuit of new
282
agonists of the protein is highly incentivized.
283
284
While largely under investigated, the use of exogenous ligands to activate STING can lead to observable
285
differences in phenotypic responses that are linked to chemical structures and protein binding properties of the
286
agonists. The most overt demonstration of this is exemplified by the human-selective agonist C53, which induces
287
activation of STING by engaging the protein’s transmembrane domain [56]. When compared with molecules
288
such as diABZI or cGAMP that activate via the cytosolic domain, C53 is unable (and indeed blocks) induction of
289
TBK1-independent STING-mediated processes such as NLRP3 inflammasome activation, autophagy, and
290
lysosomal biogenesis [7,8,57]. The collective effects of differential induction of these cellular activities on STING-
291
mediated immunological processes are virtually unknown. Additionally, while cyclic dinucleotides including
292
cGAMP, cyclic-di-AMP (CDA) and cyclic di-GMP (CDG) bind to and activate STING, they can also distinctively
293
engage STING-independent PRRs including RECON and ERAdP (CDA [58,59]), or TPL2, DDX41, and TLR7
294
(CDG [60–62]). These proteins are known to elicit innate immune effects that can enhance or counteract
295
canonical STING-mediated molecular processes through transcription factors such as IRF3 or NF-B or even
296
induce STING-independent activities such as CREB-dependent transcription. Whether the various classes of
297
small molecule agonists such as those examined here are also capable of activating STING-independent PRRs
298
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13
is unknown. In vivo, STING agonist types display differences with respect to cytokine secretion [35,63],
299
enhancement of humoral immunity to subunit vaccines [35,64,65], and cell-mediated tumor clearance [9].
300
However, an understanding of the combined mechanistic impact of differential STING engagement or induction
301
of other PRRs by STING agonists on downstream in vivo immune effects is lacking. As such, studies that
302
comparatively characterize these are necessary to inform future work whose aim is to apply a pharmacologic
303
STING agonism approach toward achieving specific clinical goals.
304
305
Work described here demonstrates measurable differences in responses to chemically different STING agonists
306
using in vitro, ex vivo, and in vivo models. This includes profound differences in transcriptional activity, cytokine
307
secretion profiles, expression of DC maturation markers, elicitation of antigen-directed humoral responses, and
308
cell mediated immune responses. Pharmacokinetic variability is certain to exist between the agonists but given
309
the differences in observed effects in vitro and ex vivo are unlikely to be solely responsible for the differences
310
detected in vivo. This could be due to engagement of STING-independent PRRs, differential induction of STING-
311
mediated processes linked to ligand-induced conformational changes, specific regions of STING binding, or
312
regulation of unknown signaling pathways. These results clearly show that DMXAA generates the most unusual
313
molecular responses despite not displaying atypical relative potency. Interestingly, DMXAA is the only agonist
314
examined here that is species selective, being inactive against primate STING orthologs [12,66]. As such it may
315
engage the protein with unique affinity or kinetics that influence activation properties. Since mammalian STING
316
evolves rapidly under positive selection, likely imposed by the fitness impacts of microbial pathogens [67],
317
species selective agonists are common [13,47,56,68–70]. Moreover, cross-reactive agonists may elicit ortholog-
318
specific molecular activities and thus their use necessitates confirming activation of immunologically relevant
319
phenotypes in both species.
320
321
These results also suggest agonist-associated differences in the ability to augment antigen-directed CD4+ and
322
CD8+ T cell responses with CDA displaying more efficacy in this regard than the small molecule agonists (Figure
323
7). This has relevance for both vaccine enhancement and anti-cancer activity as T cells play key roles in both
324
pathogen clearance and tumor cell killing. Importantly, CDA was observed to induce T cell polyfunctionality as
325
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14
indicated by their expression of both IFN and TNF. As such, better protective immune effects may be
326
demonstrable when using cyclic dinucleotides to potentiate antigen-directed immune responses. Whether this
327
can be mechanistically linked to differential molecular processes induced (or unaffected) by CDA such as
328
activation of NF-B-dependent transcription (Figure 1C), inflammasome activation (Figure 1D), or
329
transcriptomic patterns (Figure 4) requires additional inquiry but may allow discovery of precise phenotypes
330
desirable for future adjuvant development.
331
332
STING represents a valuable pharmacologic target with demonstrated beneficial applications to vaccine
333
enhancement, anti-tumor responses, antiviral protection, and pain suppression. By demonstrating agonist-
334
associated differences in molecular, cellular, and immunological responses this work provides a rationale for
335
pursuing deeper comparative characterization of STING agonists under consideration for clinical use. This
336
includes quantifying cellular processes they induce such as autophagy [6] , lysosomal biogenesis [7] , and
337
inflammasome activation [5] . Additionally, an understanding cell type-specific differences including agonist
338
sensitivity, transcriptomes, and impact on effector phenotypes will be essential to predict whether the mechanism
339
of action necessary for a desired outcome will be operational across species for a specific agonist. This will also
340
allow identification of criteria essential for validation in both animal and human models that will direct their clinical
341
use.
342
343
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15
MATERIALS AND METHODS
345
Reagents and Antibodies
346
diABZI was purchased from MedChemExpress (Cat # HY-112921B). DMXAA was purchased from ApexBio (Cat
347
# A8233). Sendai virus (SeV) was purchased from Charles River Laboratories (Cat # PI-1). Endotoxin-free
348
ovalbumin was purchased from Invivogen (Cat # vac-pova-100). Steady GLO luciferase reagent was purchased
349
from Promega (Cat # E25100). Quanti-Luc luciferase detection system was purchased from Invivogen (Cat #
350
rep-qlc4lg1). Quanti-Blue SEAP detection reagent was purchased from InVivogen (Cat # rep-qbs). Cell Titer Glo
351
was purchased from Promega (Cat # G7572). Alhydrogel aluminum hydroxide was purchased from Invivogen
352
(Cat # vac-alu-250). ML-RR-S2 CDA was purchased from Invivogen (Cat # tlrl-nacda2r-05). LPS was purchased
353
from Sigma-Aldrich (Cat # L5293). J774-Dual cells were purchased from Invivogen (cat # j774d-nfis). RAW264.7-
354
ISG-Lucia cells were purchased from Invivogen (cat # rawl-isg). GM-CSF and IL-4 were obtained from Peprotech
355
(Cat # 214-14, 315-03. Antibodies used are as follows: anti-mouse CD3 (BioLegend Cat # 100355), anti-mouse
356
CD40 (BioLegend Cat # 124622), anti-mouse CD80 (BioLegend Cat # 124622), anti-mouse CD86 (BioLegend
357
Cat # 105008), anti-mouse CD11c (BioLegend Cat # 117353), anti-mouse CD8 (Thermo Fisher Cat # 45-0081-
358
82), anti-mouse CD19 (BioLegend Cat # 115540), anti-mouse CD45R (BD Bioscience Cat # 553088), anti-
359
mouse CD11b (Thermo Fisher Cat # 48-0112-82), anti-mouse Ly-6G (BD Biosciences Cat # 562700), anti-
360
mouse F4/80 (BioLegend Cat # 123118), anti-mouse IFN (BD Biosciences Cat # 554412), anti-mouse TNF
361
(BioLegend Cat # 506307), anti-mouse CD25 (BD Biosciences Cat #564458), anti-mouse PD-1 (BioLegend Cat
362
#135225), anti-mouse OX40 (BioLegend Cat #119415), anti-mouse Bcl-6 (BD Biosciences Cat # 563363), anti-
363
mouse CD16/CD32 Ab (BD Biosciences Cat #553142), anti-mouse CD45R (BioLegend Cat #103243), anti-
364
mouse CXCR5 (BioLegend Cat #145512), anti-mouse CD3 (BioLegend Cat #100353), anti-mouse CD4 (Thermo
365
Fisher Cat #48-0041-82), and anti-mouse CD8a (Thermo Fisher Cat #MA5-17597).
366
367
Mouse Experiments
368
All mouse experiments were performed at Oregon Health and Science University in ABSL2 laboratories in
369
compliance with OHSU Institutional Animal Care and Use Committee (IACUC), under protocol 0913. The OHSU
370
IACUC adheres to NIH Office of Laboratory Animal Welfare standards (OLAW welfare assurance A3304-1).
371
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C57B6/J or IFNAR-/- mice (aged 5-8 weeks; Jackson Laboratories) were housed in ventilated cage units and
372
cared for under USDA guidelines for laboratory animals. For serum cytokine measurement, transcriptomic, and
373
dLN cell population experiments STING agonists and adjuvant controls were administered intramuscularly (IM)
374
in the quadriceps with DMXAA at 25 mg/kg, diABZI at 1.5 mg/kg, ML-RR-S2 at 0.5 mg/kg, aluminum hydroxide
375
Alum at 15 mg/kg, or PBS. For vaccination experiments animals were primed and identically boosted at 2 w and
376
harvested at 4 w (for antibody readout) or 5 w (for T cell readout). For transcriptomic and draining lymph node
377
experiments STING agonists and other adjuvants were admixed with 10 µg Endofit-ovalbumin (OVA). For T cell
378
proliferation experiments adjuvants were administered with 50 µg Endofit-OVA.
379
380
RAW264.7 and J774 Assays
381
RAW264.7-ISG-Lucia or J774-Dual cells were seeded to confluence in a 96-well plate overnight in DMEM + 10%
382
FCS. Cells were treated in duplicate with serial dilutions of STING ligands; diABZI and DMXAA starting at 100
383
µM concentrations. Lipofectamine 3000 (Invitrogen) was used to transfect CDA into RAW264.7 and J774 cells,
384
starting at 100 µM. Lipofectamine was added prior to serial dilutions of CDA. Cells were incubated with STING
385
ligands overnight, 18 h, in 50 uL of DMEM + 2% FBS. Quanti-Luc, Quanti-Blue, or Cell Titer Glo reagents were
386
added (1:1 [v/v]) to each well, and luminescence or absorbance measured on a Synergy plate reader (BioTek).
387
For IL-1 secretion experiments J774 cells were primed 6 h with 100 ng/mL LPS and treated overnight with
388
indicated stimuli. Media was harvested and IL-1 quantified using bead-based Luminex assay per the
389
manufacturer’s instructions (Thermo Fisher Cat # EPX01A-26002-901).
390
391
ELISA Assays
392
Serological responses specific to OVA were quantified by enzyme-linked immunosorbent assays (ELISA). Blood
393
was collected at 2 w post boost, stored for approx. 1 h at 4° C, and then centrifuged to separate sera. Sera was
394
aspirated and stored at -80 °C until analysis. High-binding microtiter plates were coated with OVA (1 µg/well)
395
(Invivogen) overnight at at 4° C. Coated plates were blocked using 5% (w/v) nonfat dry milk in PBS plus 0.05%
396
Tween 20 (v/v) (PBS-T) for 1 hour at room temperature. Mouse sera was heat-inactivated for 30 mins at 55°C,
397
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17
then serial diluted in 5% nonfat dry milk starting at 1:50 dilution and added to microtiter plate in duplicate. After
398
2 h incubation plates were washed 3X using accuWash plate washer (Fisher Scientific). Goat anti-mouse IgG-
399
HRP (SouthernBiotech Cat # 1030-05), anti-mouse IgG1-HRP (SouthernBiotech Cat # 1070-05), or anti-mouse
400
IgG2c-HRP (SouthernBiotech Cat # 1079-05) conjugated antibodies (at 1:10,000) were added and incubated for
401
1 h at room temp. ELISA plates were washed and developed with 0.4 mg/ml o-phenylene diamine buffer (50 nM
402
citric acid, 100 mM dibasic sodium phosphate, pH 5.0). The reaction was halted with 1 N HCl and A490 was
403
measured on a BioTek Synergy plate reader. Absorbance was quantified by calculating area under the curve
404
across serial dilutions above baseline detection.
405
406
Cytokine quantification
407
Proinflammatory cytokines in the sera were examined 5 h after injection. Blood was collected, stored for approx.
408
1 h at 4°C, and then centrifuged to separate sera which was aliquoted and stored at -80 °C until analysis. Per
409
the manufacturer’s instructions cyto/chemokines from 50 µL sample of each serum aliquot was quantified using
410
a ProcartaPlex Mouse Th1/Th2 multiplex panel (ThermoFisher Cat # EPX260-26088-901).
411
412
RNA isolation and dLN RT-qPCR
413
Total RNA was isolated from inguinal draining lymph nodes at 5, 24, and 72 hours after IM injection. RNA was
414
treated on column with DNase provided in a Quick RNA miniprep kit (Zymo Research Cat # R1055), according
415
to manufacturer’s protocol. Single-stranded cDNA was generated from total RNA using a RevertAid First Strand
416
cDNA synthesis kit (Thermo Fisher Cat # 1622), using random hexamers to prime first strand synthesis. mRNA
417
expression levels between samples was performed using semiquantitative real-time revere transcription-PCR
418
(qPCR) and quantified by ΔΔCT [71] . Prevalidated Prime-Time 6-carboxyfluorescein qPCR primer/probe sets
419
were obtained from IDT and used for all genes.
420
421
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Hybridization Array Analysis
422
Differential gene expression in lymph nodes of STING agonist IM-injected mice was determined using RNA
423
isolated from the inguinal dLN at 5 and 24 h. Hybridization array data were collected by the OHSU Integrated
424
Genomics Laboratory. RNA sample quantity and purity were measured by UV absorbance at 260, 280, and 230
425
nm with a NanoDrop 1000 spectrophotometer (ThermoScientific). RNA integrity and size distribution were
426
determined by running total RNA on a Nano chip instrument (Agilent Technologies). RNA was prepared for array
427
hybridization by labeling 100 ng aliquots using the 3’IVT Express kit (Affymetrix). RNA was reverse transcribed
428
to generate first-strand cDNA containing a T7 promoter sequence. A second-strand cDNA synthesis step was
429
performed that converted the single-stranded cDNA into a dsDNA template for transcription. Amplified and biotin-
430
labeled cRNA was generated during the in vitro transcription step. After a magnetic bead purification step to
431
remove enzymes, salts, and unincorporated nucleotides, the cRNA was fragmented. Labeled and fragmented
432
cRNA was combined with hybridization cocktail components and hybridization controls, and 130 µL of each
433
hybridization cocktail containing 6.5 µg of labeled target was injected into a cartridge containing the Clariom S
434
murine array (ThermoFisher cat # 902930) containing >221,900 probes interrogating over 150,000 transcripts
435
from >22,100 genes. Arrays were incubated for 18 h at 45°C, followed by washing and staining on a GeneChip
436
Fluidics Station 450 (Affymetrix) and the associated hybridization wash and stain kit. Arrays were scanned using
437
the GeneChip Scanner 3000 7G with an autoloader. Image inspection was performed manually immediately
438
following each scan. Image processing of sample .DAT files to generate probe intensity .CEL files was performed
439
using the Affymetrix GeneChip Command Console (AGCC) software. Each array file was then analyzed using
440
Transcriptome Analysis Console (TAC; Version 4.0.3) to obtain array performance metrics, calculate transcript
441
fold changes, and perform principal component analysis. To identify probe sets that were significantly regulated
442
in treated versus untreated (mock) cells, we employed a traditional unpaired one-way (single-factor) ANOVA for
443
each pair of condition groups as implemented in TAC. Probe sets were considered differentially regulated if the
444
ANOVA P value was <0.05 and fold change >2. Pathway analysis of significantly regulated mRNAs performed
445
using Ingenuity Pathway Analysis (Qiagen). Venn diagrams were constructed using BioVenn [72].
446
447
Intracellular Cytokine Staining (ICS) assay
448
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19
Splenocytes from vaccinated mice were incubated with 1 µg/mL OVA peptide pool (#130-099-771; Miltenyi
449
Biotec) at 37oC with 5% CO2 for 6 h in the presence of Brefeldin A (#TNB-4506; Tonbo Bioscience) in 96-well U-
450
bottom plates at a concentration of 106 cells/well. Stimulation with 25 ng/mL PMA (Invivogen Cat # tlrl-pma) and
451
500 ng/mL Ionomycin (Sigma-Aldrich Cat #10634) was included as a positive control. DMSO was used as a
452
negative control. After stimulation, the cells were incubated in anti-mouse CD16/CD32 and stained for 30 min at
453
4oC with the surface-staining fluorochrome-conjugated Abs: CD3, CD4 (Clone: GK1.5; #48-0041-82; Thermo
454
Fisher Scientific), and CD8a (Clone: CT-CD8a; # MA5-17597; Thermo Fisher Scientific). Intracellular staining
455
was performed for 30 min at 4◦C with the fluorochrome-conjugated anti-mouse IFNγ and anti-mouse TNFα using
456
BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences Cat #554714), accordingly to manufacturer’s
457
instructions. Cell events were collected on BD Symphony flow cytometer, and data were analyzed using FlowJo
458
10 software.
459
460
Activation induced marker (AIM) assay
461
Splenocytes from vaccinated mice were incubated with 2 µg/mL OVA peptide pool (Miltenyi Cat # #130-099-
462
771) at 37◦C with 5% CO2 for 18h. Stimulation with 5 µg/mL Con A (InVivogen Cat # inh-cona-2) was included
463
as a positive control. After stimulation, cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit
464
(ThermoFisher Scientific Cat # L10119) and blocked with mouse Fc block CD16/CD32 (BD Biosciences Cat
465
#553141). Cells were then stained for 30 min at 4◦C with the following fluorochrome-conjugated anti-mouse Abs:
466
CD3, CD4, CD45R/B220, CD25, PD-1, OX40, and CXCR5. Cell events were collected on LSR-II (BD
467
Biosciences) flow cytometer, and data were analyzed using FlowJo 10 software.
468
469
BMDC activation
470
Bone marrow extraction was performed by flushing RPMI media through mouse femurs. Cells were pelleted by
471
centrifugation for 5 mins at 500 x g and RBC lysed with ACK lysis (Gibco) buffer at room temp for 3 mins. After
472
centrifugation cells were resuspened in RPMI (1 mL/femur), and counted by trypan blue exclusion (Countess,
473
Invitrogen). Cells were adjusted to 5 x 106 cells per 10 cm petri dish, RPMI was supplemented with 20 ng/mL
474
GM-CSF and 10 ng/ml IL-4 for dendritic cell differentiation. Plates were incubated for 8 d with fresh supplemented
475
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media added on days 3 and 6. On day 8 adherent bone marrow derived dendritic cells (BMDC) were chemically
476
lifted from petri dish (Cellstripper, Corning Cat # 25-056-Cl). BMDCs were again counted and plated into 6-well
477
plates at 1-2 x 106 cells/well. Stimuli were added to wells as follows; CDA (1.36 µM), diABZI (17.5 nM), DMXAA
478
(17.5 µM), Alum (3 µg/ml), LPS (100 ng/ml), and DMSO (0.5%). BMDCs were incubated for 20 h, then harvested
479
as before. Antibody staining was performed in FACS buffer [PBS, 0.5% BSA (w/v), 1% 0.5 M EDTA (v/v)]. Fc-
480
mediated binding was blocked with mouse FC-Block (CD16/CD32) (BD Bioscience Cat # 553141), and dead
481
cells stained with Live/Dead Near IR. BMDCs were stained for activation markers CD80, CD86, CD40, and MHC-
482
II for 45 mins on ice. Data acquisition was performed using an LSR-II (BD Biosciences) and analyzed using
483
Flowjo 10.
484
485
APC recruitment
486
Mice were injected with STING agonists and OVA as described above. Mice euthanized at 24 h time point, and
487
inguinal dLN harvested. Single-cell suspensions were blocked with Fc Block and stained with anti-CD19, anti-
488
B220, anti-CD11b, anti-CD11c, anti-Ly6-G PE-CF594, and anti-F4/80 for 45 mins on ice. Data acquisition was
489
performed using an LSR-II (BD Biosciences) and analyzed using Flowjo 10.
490
491
ELISpot Assays
492
IFN ELISpot assays were conducted as described [73]. Single-cell suspensions of splenocytes were obtained
493
following RBC lysis and added to prewashed mouse IFNγ ELISPot plates (MabTech Cat # 3321-2A).
494
Splenocytes were stimulated with SIINFEKL peptide or OVA peptide pool (10 µg/well), Concanavalin A (0.1
495
µg/well), PMA/Ion (0.8ng/30ng/well), or 0.5% DMSO. Splenocytes were incubated in ELISpot plates for 18 h.
496
ELISpots were stained according to manufacturer’s protocol. Briefly, plates were washed and incubated with
497
anti-mouse IFNγ biotin antibody for 2 h. Steptavidin-ALP secondary antibody was added for 1 h after plates were
498
washed. Spots were detected using BCIP/NPT-plus substrate, rinsed with water, and dried before counting with
499
an AID ELISpot plate reader.
500
501
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21
FIGURE LEGENDS
502
Figure 1. Dose-dependent innate responses induced by STING agonists in murine cell lines. RAW264.7
503
(A) and J774 (B, C, D) were grown to confluence in 96 well plates and exposed in duplicate overnight to CDA,
504
DMXAA, or diABZI at indicated molarities. A. Cells were exposed to either Quanti-Luc or Cell Titer Glo lysis
505
buffer to measure IFN-I signaling induction or cell viability, respectively. Data presented are mean ± SEM relative
506
luminescence units (RLU, black) or percent viability (red) determined relative to control cells treated with DMSO-
507
or transfection reagent (CDA); C. NF-kB-dependent SEAP expression expressed as mean ± SEM absorbance
508
at OD655 for indicated treatment relative to control treated cells; D. Secretion of IL-1b from primed J774 cells
509
treated with indicated doses of DMXAA, diABZI, or CDA. Data displayed are mean ± SEM IL-1b pg/mL secreted
510
into culture media. Two-way ANOVA with Tukey’s correction for multiple comparisons was used to examine
511
statistical significance of dose-specific activation of NF-kB or IL-1b secretion between agonists (*p < 0.05, **p <
512
0.01, ***p < 0.001, ****p < 0.0001 color coded as indicated for agonist pairing).
513
514
Figure 2. Maturation of Dendritic Cells from C57Bl/6 Mice in Response to Innate Stimuli. Immature bone
515
marrow derived DC (BMDC) from C57Bl/6 mice were treated with 0.5% DMSO, 1 µM diABZI, 50 μM DMXAA, 1
516
µM CDA, 100 ng/mL LPS, or 3 µg/mL Alum as indicated. Cells were collected after 24 h and stained for
517
expression of CD40, CD80, CD86 and MHC-II and analyzed by flow cytometry. A. Mean fluorescence intensities
518
(MFI) ± SD for indicated treatment and surface marker. One way ANOVA with Tukey’s multiple comparisons test
519
was used to compare results between treatments (***p < 0.001****; p < 0.0001); B. Representative MFI
520
histograms for each marker are shown for cells exposed to indicated stimulus.
521
522
Figure 3. Induction of systemic cytokine secretion by STING agonists in vivo. C57Bl/6 mice were injected
523
IM with PBS, 30 µg diABZI, 10 µg, CDA, or 500 µg DMXAA and serum harvested at 24 h. Luminex bead based
524
multiplex assay was then used to measure absolute levels of indicated cytokines. Data presented are mean ±
525
SD pg/mL. One way ANOVA with Dunnet correction for multiple comparisons was used to determine statistical
526
significance (**p <0.01, ***p < 0.001, ***p < 0.0001).
527
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22
528
Figure 4. STING agonist-induced transcriptomic changes in vivo. C57Bl/6 mice were injected
529
intramuscularly with PBS, CDA, diABZI or DMXAA and total RNA from draining lymph nodes harvested at
530
indicated time point. A. Levels of indicated mRNA transcript in draining lymph nodes at 5 h, 24 h, or 72 h for
531
indicated treatment as determined by qPCR. Data presented are mean ± SEM fold changes relative to PBS
532
treated control animals. Statistical significance was determined using a mixed effects ANOVA with Tukey’s
533
correction for multiple comparisons of log transformed fold changes between stimuli at each time point (*p <
534
0.05; **p < 0.01); B. Number of mRNAs significantly up or downregulated more than twofold relative to PBS
535
treatment following indicated treatment as determined by hybridization array; C. Principal component analysis of
536
transcript fold changes for indicated stimulus and time point; D. Venn diagram of transcripts significantly
537
upregulated for indicated stimulus at 5 h and 24 h; E. Signaling pathways displaying a predicted Z score > 1 for
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all three stimuli at 5 h and 24 h as determined by Ingenuity Pathway Analysis; F. Pathways displaying a predicted
539
Z score > 1 following only DMXAA treatment at 5 h and 24 h.
540
541
Figure 5. STING agonist-induced changes in draining lymph node immune cell populations. In duplicate
542
experiments C57Bl/6 mice were injected intramuscularly with OVA in the presence of PBS, CDA, diABZI DMXAA,
543
or Alum and single cell suspensions of draining lymph nodes obtained at 24 h post treatment. Flow cytometry
544
was then used to quantify indicated cell types. A. Total number of lymph node cells following indicated treatment;
545
B. Percent of total lymph node cells represented by indicated B cells, T cells, cDC1, cDC2, pDC, neutrophils,
546
and macrophages as indicated; C. Percent of total lymph node cells represented by indicated B cells, T cells,
547
cDC1, cDC2, pDC, neutrophils, and macrophages in WT and IFNAR-/- mice as indicated. Statistical significance
548
was determined using ANOVA with Dunnet’s correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p <
549
0.001; ****p < 0.0001). Individual experiments indicated by circle color.
550
551
Figure 6. Enhancement of humoral immune response to OVA by co-administered STING agonists.
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C57Bl/6 mice were vaccinated IM with OVA in the presence of PBS, CDA, diABZI, DMXAA, or Alum, identically
553
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.21.639458doi: bioRxiv preprint
23
boosted 2 w later and harvested 2 w later. A. ELISA was performed using serial dilution of harvested sera to
554
quantify levels of total IgG, IgG1, and IgG2c reactive to OVA antigen. Data presented are mean + SEM
555
absorbance (top) as well as mean + SD area under curve (AUC) of absorbance signal including individual animal
556
measurements (bottom); Total number of lymph node cells following indicated treatment; B. Mean + SD ratio of
557
IgG2c to IgG1 AUC titers; C. Mean + SD percentage of OVA-reactive (OX40/CD25+) TFH (CXCR5+PD-1high)
558
cells harvested seven days after boost with OVA + indicated adjuvant. Statistical significance was determined
559
using ANOVA with Dunnet’s correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p <
560
0.0001).
561
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Figure 7. STING-adjuvant associated enhancement of cell mediated responses. C57Bl/6 mice were left
563
untreated or vaccinated IM using a prime/boost strategy with OVA in the presence of PBS, CDA, diABZI, DMXAA,
564
or Alum. A. Splenocytes were harvested at 7 d post boost and stimulated ex vivo with a peptide pool spanning
565
the OVA protein (left) or SIINFEKL peptide (right). ELISpot was then used to quantify the number of IFNg positive
566
cells. Data presented are mean ± SD number of IFNg positive spot forming units (SFU) per 2.5 x 105 total cells.
567
Results from individual animals are also presented with duplicate experiments represented by marker color; B.
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Intracellular cytokine staining and flow cytometry was used to quantify OVA peptide pool-stimulated expression
569
of IFNg and TNFa from CD4+ and CD8+ T cells in splenocytes. Data presented are mean ± SD percentage of
570
CD8+ (left) or CD4+ (right) T cells that display positivity for indicated cytokine. Individual animals represented by
571
circles. One way ANOVA using Tukey’s multiple comparison was used to determine statistical significance (*p <
572
0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
573
574
Supplemental Table 1. STING agonist-mediated dLN transcriptomic changes. Fold changes of mapped
575
transcripts from hybridization array. Data presented are log2 transformed fold changes for mRNAs and
576
corresponding probe IDs for indicated stimulus relative to mock treated animals and -log(FDRp) values.
577
578
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.21.639458doi: bioRxiv preprint
24
Supplemental Table 2. Stimulus-induced pathway regulation. Predicted Z scores of indicated biological
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pathways based on dLN transcriptomic patterns of STING agonists.
580
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Supplemental Figure 1. Representative flow cytometry gating plots for dLN B cells, T cells, neutrophils, pDC,
582
cDC1, cDC2, pDC, and macrophages.
583
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Supplemental Figure 2. Representative flow cytometry gating plots for IFN+ and TNF+ CD4+ and CD8+ T
585
cells.
586
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Supplemental Figure 3. Representative flow cytometry gating plots for activated TFH cells.
588
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.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.21.639458doi: bioRxiv preprint
25
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31
Funding 802
This research was supported by NIH R01 AI143660, R01 AI143660, and HHSN272201400055C. 803
CRediT authorship contribution statement 804
Victor R. DeFilippis: Writing – original draft, Validation, Methodology, Formal analysis, Conceptualization, 805
Supervision, Funding acquisition. Nobuyo Mizuno: Writing –Methodology, Investigation, Formal 806
analysis. Dylan Boehm: Investigation, Formal analysis. Kevin Jimenez-Perez: Investigation, Formal 807
analysis. Jinu Abraham: Investigation, Formal analysis. Laura Springgay: Investigation, Formal analysis. Ian 808
Rose: Investigation, Formal analysis. 809
Declaration of competing interest 810
The authors declare no financial interests/personal relationships which may be considered as potential 811
competing interests. 812
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A
B
Figure 1. Dose-dependent innate responses induced by STING agonists in murine cell lines. RAW264.7 (A) and J774 (B, C, D) were grown to confluence in 96 well plates and
exposed in duplicate overnight to CDA, DMXAA, or diABZI at indicated molarities. A. Cells were exposed to either Quanti-Luc or Cell Titer Glo lysis buffer to measure IFN-I signaling
induction or cell viability, respectively. Data presented are mean ± SEM relative luminescence units (RLU, black) or percent viability (red) determined relative to control cells treated with
DMSO- or transfection reagent (CDA); C. NF-B-dependent SEAP expression expressed as mean ± SEM absorbance at OD655 for indicated treatment relative to control treated cells; D.
Secretion of IL-1 from primed J774 cells treated with indicated doses of DMXAA, diABZI, or CDA. Data displayed are mean ± SEM IL-1 pg/mL secreted into culture media. Two-way
ANOVA with Tukey’s correction for multiple comparisons was used to examine statistical significance of dose-specific activation of NF-B or IL-1 secretion between agonists (*p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001 color coded as indicated for agonist pairing).
C
D
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Figure 2. Maturation of Dendritic Cells from C57Bl/6 Mice in Response to Innate Stimuli. Immature bone marrow
derived DC (BMDC) from C57Bl/6 mice were treated with 0.5% DMSO, 1 µM diABZI, 50 μM DMXAA, 1 µM CDA, 100 ng/mL
LPS, or 3 µg/mL Alum as indicated. Cells were collected after 24 h and stained for expression of CD40, CD80, CD86 and
MHC-II and analyzed by flow cytometry. A. Mean fluorescence intensities (MFI) ± SD for indicated treatment and surface
marker. One way ANOVA with Tukey’s multiple comparisons test was used to compare results between treatments (***p <
0.001****; p < 0.0001); B. Representative MFI histograms for each marker are shown for cells exposed to indicated stimulus.
B
CD40 MFI CD80 MFI CD86 MFI MHC-II MFI
DMSO
Counts
CDA
diABZI
DMXAA
LPS
Alum
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PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
PBS
diABZI
CDA
DMXAA
Figure 3. Induction of systemic cytokine secretion by STING agonists in vivo. C57Bl/6 mice were injected IM with PBS, 30 µg diABZI, 10 µg, CDA, or 500 µg DMXAA and serum
harvested at 24 h. Luminex bead based multiplex assay was then used to measure absolute levels of indicated cytokines. Data presented are mean ± SD pg/mL. One way ANOVA with
Dunnet correction for multiple comparisons was used to determine statistical significance (**p <0.01, ***p < 0.001, ***p < 0.0001).
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*
*
*
**
**
*
**
*
**
*
*
*
*
**
*
**
*
*
*
**
**
*
*
*
**
*
**
*
*
*
*
**
**
*
*
*
*
**
*
*
*
**
**
Vs.
Figure 4. STING agonist-induced transcriptomic changes in vivo. C57Bl/6 mice were injected intramuscularly with PBS, CDA, diABZI or DMXAA and total RNA from draining lymph nodes
harvested at indicated time point. A. Levels of indicated mRNA transcript in draining lymph nodes at 5 h, 24 h, or 72 h for indicated treatment as determined by qPCR. Data presented are
mean ± SD fold changes relative to PBS treated control animals. Statistical significance was determined using a mixed effects ANOVA with Tukey’s correction for multiple comparisons of log
transformed fold changes between stimuli at each time point (*p < 0.05; **p < 0.01); B. Number of mRNAs significantly up or downregulated more than twofold relative to PBS treatment
following indicated treatment as determined by hybridization array; C. Principal components analysis of transcript fold changes for indicated stimulus and time point; D. Venn diagram of
transcripts significantly upregulated for indicated stimulus at 5 h and 24 h; E. Signaling pathways displaying a predicted Z score > 1 for all three stimuli at 5 h and 24 h as determined by
Ingenuity Pathway Analysis; F. Pathways displaying a predicted Z score > 1 for following DMXAA treatment only at 5 h and 24 h.
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CDA 5h
diABZI 5h
DMXAA 5h
CDA 24h
diABZI 24h
DMXAA 24h
0
1000
2000
3000
4000
5000
6000
# Transcripts
mRNA Regulation
Down
Up
CDA
diABZI
DMXAA
CDA
diABZI
DMXAA
5 h 24 h
BCD
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5 h
24 h
E
F
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PBS
CDA
diABZI
DMXAA
Alum
100
101
102
103
104
105
106
107
Total Cells
Total dLN Cells
B Cells
T Cells
cDC1
cDC2 pDC Neutrophils Macrophages
A
B
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IFNAR-/-
C57Bl/6
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
OVA
OVA + DMXAA
C
Figure 5. STING agonist-induced changes in draining lymph node immune cell populations. In duplicate experiments C57Bl/6 mice were injected intramuscularly with OVA in the
presence of PBS, CDA, diABZI DMXAA, or Alum and single cell suspensions of draining lymph nodes obtained at 24 h post treatment. Flow cytometry was then used to quantify indicated cell
types. A. Total number of lymph node cells following indicated treatment; B. Percent of total lymph node cells represented by indicated B cells, T cells, cDC1, cDC2, pDC, neutrophils, and
macrophages as indicated; C. Percent of total lymph node cells represented by indicated B cells, T cells, cDC1, cDC2, pDC, neutrophils, and macrophages in WT and IFNAR-/- mice as
indicated. Statistical significance was determined using ANOVA with Dunnet’s correction for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Individual experiments
indicated by circle color.
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PBS
CDA
diABZI
DMXAA
Anti-OVA Total IgG Anti-OVA IgG1 Anti-OVA IgG2c
Alum
Anti-OVA IgG2c
Anti-OVA IgG1
101102103104105106
0.0
0.5
1.0
1.5
2.0
Dilutions
Abs490
IgG1- Absorbance
OVA + PBS
OVA + CDA
OVA + diABZI
OVA + DMXAA
OVA + Alum
Naive
101102103104105106
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Dilutions
Abs490
IgG2c- Absorbance
OVA + PBS
OVA + CDA
OVA + diABZI
OVA + DMXAA
OVA + Alum
Naive
Anti-OVA Total IgG
IgG2c:IgG1
AUC Ratio
PBS
CDA
diABZI
DMXAA
Alum
PBS
CDA
diABZI
DMXAA
Alum
PBS
CDA
diABZI
DMXAA
Alum
A
Figure 6. Enhancement of humoral immune
response to OVA by co-administered
STING agonists. C57Bl/6 mice were
vaccinated IM with OVA in the presence of
PBS, CDA, diABZI, DMXAA, or Alum,
identically boosted 2 w later and harvested 2
w later. A. Absorbance of serum antibodies
reactive to OVA as determined by ELISA. Data
presented are mean + SEM raw 490
absorbance over indicated serum dilution for
IgG, IgG1, and IgG2c; B. Antibody titers as
determined by area under the curve (AUC)
calculations for each adjuvant. Data presented
are mean + SEM AUC including individual
animal measurements; C. Mean + SD ratio of
IgG2c to IgG1 AUC titers; D. Number of OVA-
reactive TFH (CD4+CXCR5+PD-1+) splenocytes
harvested seven days after boost with OVA +
indicated adjuvant. Statistical significance was
determined using ANOVA with Dunnet’s
correction for multiple comparisons (*p < 0.05;
**p < 0.01; ***p < 0.001; ****p < 0.0001).
Log AUC
B
C
D
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Figure 7. STING-Adjuvant Associated Enhancement of Cell Mediated Responses. C57Bl/6 mice were left untreated or vaccinated IM using a prime/boost strategy with OVA in the presence of PBS, CDA, diABZI, DMXAA, or Alum. A. Splenocytes were
harvested at 7 d post boost and stimulated ex vivo with a peptide pool spanning the OVA protein (left) or SIINFEKL peptide (right). ELISpot was then used to quantify the number of IFN positive cells. Data presented are mean ± SD number of IFN positive
spot forming units (SFU) per 2.5 x 105 total cells. Results from individual animals are also presented with duplicate experiments represented by marker color; B. Intracellular cytokine staining and flow cytometry was used to quantify OVA peptide pool-
stimulated expression of IFN and TNF from CD4+ and CD8+ T cells in splenocytes. Data presented are mean ± SD percentage of CD8+ (left) or CD4+ (right) T cells that display positivity for indicated cytokine. Individual animals represented by circles. One
way ANOVA using Tukey’s multiple comparison was used to determine statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
PBS
CDA
diABZI
DMXAA
Alum
Naive
PBS
CDA
diABZI
DMXAA
Alum
Naive
PBS
CDA
diABZI
DMXAA
Alum
Naive
PBS
CDA
diABZI
DMXAA
Alum
Naive
PBS
CDA
diABZI
DMXAA
Alum
Naive
PBS
CDA
diABZI
DMXAA
Alum
Naive
A
B
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