Stimulator Of Interferon Genes Activity Research Articles

A dual-STING-activating nanosystem expands cancer immunotherapeutic temporal window.

Stimulator of interferon genes (STING) is a promising antitumor target via bridging innate and adaptive immunity, yet the transient nature of immune signal transduction renders small-molecule agonists susceptible to short time effectiveness. Here, we report a dual-STING-activating micelle system (D-SAM) to dynamically program STING kinetics. Mechanistically, the natural ligand cGAMP encapsulated in D-SAM initiates STING signaling, while the pH-sensitive polymeric agonist PC7A disassembled from micelle shell buffers lysosomal protons and retards STING degradation. This prolonged STING activity facilitates dendritic cell (DC) antigen presentation and extends cytotoxic T lymphocyte priming. D-SAM improves efficacy over single soluble or delivered agonists against established, metastatic, and recurring murine tumors. Specific depletion of STING in DCs or blockade of CD8+ Tcell infiltration abrogates therapeutic effects. The feasibility of immune modulation is further validated in resected human patient tissues. This work underscores the temporal rhythm of STING as crucial for mounting a potent and enduring antitumor immune response.

Loss of STING impairs lactogenic differentiation.

Heightened energetic and nutrient demand during lactogenic differentiation of the mammary gland elicits upregulation of various stress responses to support cellular homeostasis. Here, we identify the stimulator of interferon genes (STING) as an immune supporter of the functional development of mouse mammary epithelial cells (MECs). An in vitro model of MEC differentiation revealed that STING is activated in a cGAS-independent manner to produce both type I interferons and proinflammatory cytokines in response to the accumulation of mitochondrial reactive oxygen species. Induction of STING activity was found to be dependent on the breast tumor suppressor gene single-minded 2 (SIM2). Using mouse models of lactation, we discovered that loss of STING activity results in early involution of #3 mammary glands, severely impairing lactational performance. Our data suggest that STING is required for successful functional differentiation of the mammary gland and bestows a differential lactogenic phenotype between #3 mammary glands and the traditionally explored inguinal 4|9 pair. These findings affirm unique development of mammary gland pairs that is essential to consider in future investigations into normal development and breast cancer initiation.

Involvement of cGAS/STING Signaling in the Pathogenesis of Candida albicans Keratitis: Insights From Genetic and Pharmacological Approaches.

Fungal keratitis (FK) is an invasive corneal infection associated with significant risk to vision. Although the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway has been recognized for its role in defending against viral infections, its involvement in FK still remains largely unclear. This study sought to elucidate the contribution of the cGAS/STING signaling pathway to the pathogenesis of FK. The expression of cGAS/STING signaling components was assessed in a murine model of Candida albicans keratitis through RNA sequencing, western blot analysis, immunofluorescence staining, and real-time PCR. Both genetic (utilizing Sting1gt/gt mice) and pharmacological (using C176) interventions were employed to inhibit STING activity, allowing for the evaluation of resultant pathogenic alterations in FK using slit-lamp examination, clinical scoring, hematoxylin and eosin (H&E) staining, fungal culture, and RNA sequencing. Subconjunctival administration of the NOD-like receptor protein 3 (NLRP3) inflammasome inhibitor MCC950 was performed to evaluate FK manifestations following STING activity blockade. Furthermore, the impact of the STING agonist diABZI on FK progression was investigated. Compared to uninfected corneas, those infected with C. albicans exhibited increased expression of cGAS/STING signaling components, as well as its elevated activity. Inhibiting cGAS/STING signaling exacerbated the advancement of FK, as evidenced by elevated clinical scores, augmented fungal load, and heightened inflammatory response, including NLRP3 inflammasome activation and pyroptosis. Pharmacological inhibition of the NLRP3 inflammasome effectively mitigated the exacerbated FK by suppressing STING activity. Conversely, pre-activation of STING exacerbated FK progression compared to the PBS control, characterized by increased fungal burden and reinforced inflammatory infiltration. This study demonstrates the essential role of the cGAS/STING signaling pathway in FK pathogenesis and highlights the necessity of its proper activation for the host against FK.

ISGylation by HERCs facilitates STING activation

Optimal activation of stimulator of interferon genes (STING) protein is crucial for host defenses against pathogens and avoiding detrimental effects. Various post-translational modifications control STING activity. However, the function of interferon (IFN)-stimulated gene (ISG) 15 modification (ISGylation) in controlling STING stability and activation is unclear. Here, we show that the E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice) facilitate STING activation by mediating ISGylation of STING at K150, preventing its K48-linked ubiquitination and degradation. Concordantly, Herc6 deficiency suppresses herpes simplex virus 1 infection-induced type I IFN responses and facilitates viral replication both invitro and invivo. Notably, severe acute respiratory syndrome coronavirus 2 protein papain-like protease cleaves HERC5-mediated ISGylation of STING, suppressing host antiviral responses. These data identify a mechanism by which HERCs-mediated ISGylation controls STING stability and activation and uncover the correlations and interactions of ISGylation and ubiquitination during STING activation.

Aryl hydrocarbon receptor suppresses STING-mediated type I IFN expression in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is one of the most aggressive types of cancer. Despite decades of intense investigation, treatment options remain limited, and rapid recurrence with distant metastases remains a significant challenge. Cancer cell-intrinsic production of cytokines such as type I interferons (IFN-I) is a known potent modulator of response to therapy in many cancers, including TNBC, and can influence therapeutic outcome. Here, we report that, in TNBC systems, the aryl hydrocarbon receptor (AhR) suppresses IFN-I expression via inhibition of STImulator of Interferon Genes (STING), a key mediator of interferon production. Intratumoral STING activity is essential in mediating the efficacy of PARP inhibitors (PARPi) which are used in the treatment of cancers harboring BRCA1 deficiency. We find that, in TNBC cells, PARPi treatment activates AhR in a BRCA1 deficiency-dependent manner, thus suggesting the presence of a negative feedback loop aimed at modulating PARPi efficacy. Importantly, our results indicate that the combined inhibition of PARP and AhR is superior in elevating IFN-I expression as compared to PARPi-alone. Thus, AhR inhibition may allow for enhanced IFN-I production upon PARPi in BRCA1-deficient breast cancers, most of which are of TNBC origin, and may represent a therapeutically viable strategy to enhance PARPi efficacy.

Neuregulin-1, a member of the epidermal growth factor family, mitigates STING-mediated pyroptosis and necroptosis in ischaemic flaps

Abstract Background Ensuring the survival of the distal end of a random flap during hypoperfusion (ischaemia) is difficult in clinical practice. Effective prevention of programmed cell death is a potential strategy for inhibiting ischaemic flap necrosis. The activation of stimulator of interferon genes (STING) pathway promotes inflammation and leads to cell death. The epidermal growth factor family member neuregulin-1 (NRG1) reduces cell death by activating the protein kinase B (AKT) signalling pathway. Moreover, AKT signalling negatively regulates STING activity. We aimed to verify the efficacy of NRG1 injection in protecting against flap necrosis. Additionally, we investigated whether NRG1 effectively enhances ischemic flap survival by inhibiting pyroptosis and necroptosis through STING suppression. Methods A random-pattern skin flap model was generated on the backs of C57BL/6 mice. The skin flap survival area was determined. The blood supply and vascular network of the flap was assessed by laser Doppler blood flow analysis. Cluster of differentiation 34 immunohistochemistry (IHC) and haematoxylin and eosin (H&E) staining of the flap sections revealed microvessels. Transcriptome sequencing analysis revealed the mechanism by which NRG1 promotes the survival of ischaemic flaps. The levels of angiogenesis, oxidative stress, necroptosis, pyroptosis and indicators associated with signalling pathways in flaps were examined by IHC, immunofluorescence and Western blotting. Packaging adeno-associated virus (AAV) was used to activate STING in flaps. Results NRG1 promoted the survival of ischaemic flaps. An increased subcutaneous vascular network and neovascularization were found in ischaemic flaps after the application of NRG1. Transcriptomic gene ontology enrichment analysis and protein level detection indicated that necroptosis, pyroptosis and STING activity were reduced in the NRG1 group. The phosphorylation of AKT and forkhead box O3a (FOXO3a) were increased after NRG1 treatment. The increased expression of STING in flaps induced by AAV reversed the therapeutic effect of NRG1. The ability of NRG1 to phosphorylate AKT-FOXO3a, inhibit STING and promote flap survival was abolished after the application of the AKT inhibitor MK2206. Conclusions NRG1 inhibits pyroptosis and necroptosis by activating the AKT-FOXO3a signalling pathway to suppress STING activation and promote ischaemic flap survival.

Identification of (−)-Epigallocateshin gallate derivatives promoting innate immune activation via 2′,3′-cyclic GMP-AMP-stimulator of interferon genes pathway

(−)-Epigallocatehin-3-gallate (EGCG) is a catechin derived from green tea, which has been widely studied for its anti-oxidant and anti-tumor properties. Although EGCG plays important roles in various biological processes, the its effect on the immune system is not fully understood. In this study, we investigated the potential of EGCG as an activator of the stimulator of interferon genes (STING) pathway in the immune system. The cyclic GMP-AMP synthase (cGAS)-2-3-cyclic GMP-AMP (cGAMP)-STING pathway is crucial in the innate immune response to microbial infections, autoimmunity, and anticancer immunity. We confirmed that EGCG enhanced the immune response of cGAMP and identified E2 from 13 synthetic derivatives of EGCG. E2 specifically activated the interferon (IFN) signaling pathway specifically through STING- and cGAMP-dependent mechanisms. These results demonstrate the potential of EGCG and its derivatives as new STING activators that can stimulate the type I interferon response by boosting cGAMP-mediated STING activity.

STING-dependent interferon signatures restrict osteoclast differentiation and bone loss in mice

Stimulator of interferon genes (STING) is a key mediator of type-I interferon (IFN-I) signaling in response to a variety of stimuli, but the contribution of STING to homeostatic processes is not fully characterized. Previous studies showed that ligand activation of STING limits osteoclast differentiation invitro through the induction of IFNβ and IFN-I interferon-stimulated genes (ISGs). In a disease model (SAVI) driven by the V154M gain-of-function mutation in STING, fewer osteoclasts form from SAVI precursors in response to receptor activator of NF-kappaB ligand (RANKL) in an IFN-I-dependent manner. Due to the described role of STING-mediated regulation of osteoclastogenesis in activation settings, we sought to determine whether basal STING signaling contributes to bone homeostasis, an unexplored area. Using whole-body and myeloid-specific deficiency, we show that STING signaling prevents trabecular bone loss in mice over time and that myeloid-restricted STING activity is sufficient for this effect. STING-deficient osteoclast precursors differentiate with greater efficiency than wild types. RNA sequencing of wild-type and STING-deficient osteoclast precursor cells and differentiating osteoclasts reveals unique clusters of ISGs including a previously undescribed ISG set expressed in RANKL naïve precursors (tonic expression) and down-regulated during differentiation. We identify a 50 gene tonic ISG signature that is STING dependent and shapes osteoclast differentiation. From this list, we identify interferon-stimulated gene 15 (ISG15) as a tonic STING-regulated ISG that limits osteoclast formation. Thus, STING is an important upstream regulator of tonic IFN-I signatures shaping the commitment to osteoclast fates, providing evidence for a nuanced and unique role for this pathway in bone homeostasis.

STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma.

Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1-6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFβ. STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner-these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse.

STAM transports STING oligomers into extracellular vesicles, down-regulating the innate immune response.

Stimulator of interferon genes (STING) mediates the innate immune response against damaged endogenous double-strand DNA and exogenous virus infection. The location of STING is critical to the accurate control of defence signalling pathways. Recently, the effects of extracellular vesicles (EVs) in the regulation of innate immune signalling have been reported. Nevertheless, the particular roles played by STING in EVs and the related mechanisms have remained largely unknown. Herein, we report that when STING was activated in cells, EVs derived from these cells carried STING oligomers. Signal transducing adapter molecule 1 (STAM) was found to be a STING transporter that directly interacted with STING and facilitated STING transport into EVs. Importantly, the translocation of STING into EVs was a mechanism by which STING was degraded, suppressing the innate immune response. In summary, we elucidated the mechanism and function of the translocation of STING into EVs, adding to the understanding of STING activity regulation.

Immunogenicity of small-cell lung cancer associates with STING pathway activation and is enhanced by ATR and TOP1 inhibition.

The activation of STING (stimulator of interferon genes) pathway enhances antitumor immunity in small-cell lung cancer (SCLC), while the DNA damage induced by non-cGAMP-based agonists is a potent inducer of STING activity. Here, we investigate the intrinsic expression of STING in cancer cells and evaluate the value of the combination of ATR and TOP1 inhibitors in enhancing antitumor immunity. STING expression was assessed at mRNA and protein levels in SCLC and normal lung tissues. Transcriptomic subsets of SCLC were identified based on STING-related genes. Distinct mutation and immunogenomic profiles of these subsets were determined. The direct antitumor efficacy and the potential of enhancing antitumor immunity of the strategy using the ATR-TOP1-inhibitor combination were tested in SCLC cell lines. The intrinsic expression of STING was significantly reduced in SCLC compared to normal lung tissues (p < 0.0001). Three STING-related SCLC subtypes were identified in which the STING-high subtype was associated with (1) high immune infiltration, (2) high expression of genes related to MHC and immune checkpoints, and (3) high EMT and ferroptosis score. On the contrary, the STING-low subtype was enriched with pathways related to DNA damage response (DDR) and cell cycle progression. The association between the DDR pathway activity and the STING-IFN innate immune response was verified by in vitro experiments in which the inhibition of ATR and TOP1 triggered the expression of genes encoding type I IFN signaling and pro-inflammatory cytokines/chemokines in a STING-low SCLC cell line. Our study verifies that activation of the STING-IFN response by ATR and TOP1 inhibitors might be a therapeutic strategy to improve the response to immune checkpoint therapy in STING-low SCLC. Furthermore, the combinations of ATR and TOP1 inhibitors can augment tumor inflammation in STING-low SCLC.

How Fragile We Are: Influence of Stimulator of Interferon Genes (STING) Variants on Pathogen Recognition and Immune Response Efficiency.

The stimulator of interferon genes (STING) protein is a cornerstone of the human immune response. Its activation by cGAMP in the presence of cytosolic DNA stimulates the production of type I interferons and inflammatory cytokines. In the human population, several STING variants exist and exhibit dramatic differences in their activity, impacting the efficiency of the host defense against infections. Understanding the molecular mechanisms of these variants opens perspectives for personalized medicine treatments against diseases such as viral infections, cancers, or autoinflammatory diseases. Through microsecond-scale molecular modeling simulations, contact analyses, and machine learning techniques, we reveal the dynamic behavior of four STING variants (wild type, G230A, R293Q, and G230A/R293Q) and rationalize the variability of efficiency observed experimentally. Our results show that the decrease in STING activity is linked to a stiffening of key structural elements of the binding cavity together with changes in the interaction patterns within the protein.

Sting Is Commonly and Differentially Expressed in T- and Nk-Cell but Not B-Cell Non-Hodgkin Lymphomas.

Simple SummaryT/NK-cell non-Hodgkin lymphomas (NHLs) represent approximately 10% of all NHLs and most patients have a poor outcome using current treatment options. Molecules involved in the host response against lymphoma cells are currently being investigated in an effort to develop novel therapeutic strategies combining targeted therapy and immunotherapy. In this study, we show that expression of STING, a key protein in the cGAS–STING immune response pathway, is restricted to lymphomas of T- and NK-cell origin and seems to be down regulated in B-cell NHLs. These results are based on the analysis of 14 lymphoma cell lines of various types at the RNA and protein level and immunohistochemical analysis of a large number of B-cell (n = 265) and T/NK-cell (n = 158) NHLs obtained from previously untreated patients from three institutions. In these patient cohorts, STING is differentially expressed among T/NK-cell NHLs, whereas all B-cell NHLs were negative for STING expression. Thus, STING represents a novel biomarker and therapeutic target in T- and NK-cell lymphomas with direct immunotherapeutic implications, since modulators of cGAS–STING activity are already available for clinical use, and could therefore be used to benefit patients with these difficult-to-treat diseases. The expression patterns of stimulator of interferon genes (STING) were investigated in a cohort of 158 T- and natural killer (NK)-cell and 265 B-cell non-Hodgkin lymphomas (NHLs), as well as in control reactive lymph nodes and tonsils. STING expression was assessed by immunohistochemical methods using diagnostic biopsy specimens obtained prior to treatment. Using an arbitrary 10% cutoff, STING was differentially expressed among T/NK-cell NHLs; positive in 36 out of 38 (95%) cases of ALK+ anaplastic large cell lymphoma (ALCL), 23 out of 37 (62%) ALK-ALCLs, 1 out of 13 (7.7%) angioimmunoblastic T-cell lymphomas, 15 out of 19 (79%) peripheral T-cell lymphomas, not otherwise specified, 20 out of 36 (56%) extranodal NK/T-cell lymphomas of nasal type, 6 out of 7 (86%) T-cell lymphoblastic lymphomas, and 3 out of 4 (75%) mycosis fungoides. STING expression did not correlate with clinicopathological parameters or outcome in these patients with T/NK-cell lymphoma. By contrast, all 265 B-cell NHLs of various types were STING-negative. In addition, STING mRNA levels were very high in 6 out of 7 T-cell NHL cell lines, namely, ALK+ and ALK-ALCL cell lines, and very low or undetectable in 7 B-cell NHL cell lines, suggesting transcriptional downregulation of STING in neoplastic B-cells. At the protein level, using Western blot analysis and immunohistochemistry performed on cell blocks, STING expression was found to be restricted to T-cell NHL cell lines. Taken together, STING expression represents a novel biomarker and therapeutic target in T- and NK-cell lymphomas with direct immunotherapeutic implications since modulators of cGAS–STING activity are already available for clinical use.

The cGAS\u2013STING pathway drives type I IFN immunopathology in COVID-19

COVID-19, which is caused by infection with SARS-CoV-2, is characterized by lung pathology and extrapulmonary complications1,2. Type I interferons (IFNs) have an essential role in the pathogenesis of COVID-19 (refs 3–5). Although rapid induction of type I IFNs limits virus propagation, a sustained increase in the levels of type I IFNs in the late phase of the infection is associated with aberrant inflammation and poor clinical outcome5–17. Here we show that the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway, which controls immunity to cytosolic DNA, is a critical driver of aberrant type I IFN responses in COVID-19 (ref. 18). Profiling COVID-19 skin manifestations, we uncover a STING-dependent type I IFN signature that is primarily mediated by macrophages adjacent to areas of endothelial cell damage. Moreover, cGAS–STING activity was detected in lung samples from patients with COVID-19 with prominent tissue destruction, and was associated with type I IFN responses. A lung-on-chip model revealed that, in addition to macrophages, infection with SARS-CoV-2 activates cGAS–STING signalling in endothelial cells through mitochondrial DNA release, which leads to cell death and type I IFN production. In mice, pharmacological inhibition of STING reduces severe lung inflammation induced by SARS-CoV-2 and improves disease outcome. Collectively, our study establishes a mechanistic basis of pathological type I IFN responses in COVID-19 and reveals a principle for the development of host-directed therapeutics.

Human STING Is Regulated by an Autoinhibitory Mechanism for Type I Interferon Production

Stimulator of interferon genes (STING) plays a pivotal role in type I interferon-mediated innate immune response to the cytoplasmic detection of aberrant DNA. STING is a membrane protein localized in endoplasmic reticulum (ER), which upon stimulation translocates to Golgi apparatus and activates downstream signaling cascades. However, the mechanism regulating STING activity and significance of its intracellular traffic are not completely understood. Here we identify a novel region of human STING comprising thirteen residues within its C-terminal tail (CTT) for downstream nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation. We also discover that STING CTT fragment can activate downstream signaling regardless of its ER localization. In addition, we reveal that ligand-binding domain (LBD) in the middle of STING binds and confers autoinhibition to its CTT for both NF-κB- and interferon regulatory factor 3-activation. Furthermore, STING LBD can inhibit the interferon-stimulating activity of STING CTT in trans and demonstrate a dominant negative effect on endogenous STING for interferon induction. We thus uncover an important autoinhibitory mechanism modulating STING activity.

Development of Small-Molecule STING Activators for Cancer Immunotherapy

In cancer immunotherapy, the cyclic GMP–AMP synthase–stimulator of interferon genes (STING) pathway is an attractive target for switching the tumor immunophenotype from ‘cold’ to ‘hot’ through the activation of the type I interferon response. To develop a new chemical entity for STING activator to improve cyclic GMP-AMP (cGAMP)-induced innate immune response, we identified KAS-08 via the structural modification of DW2282, which was previously reported as an anti-cancer agent with an unknown mechanism. Further investigation revealed that direct STING binding or the enhanced phosphorylation of STING and downstream effectors were responsible for DW2282-or KAS-08-mediated STING activity. Furthermore, KAS-08 was validated as an effective STING pathway activator in vitro and in vivo. The synergistic effect of cGAMP-mediated immunity and efficient anti-cancer effects successfully demonstrated the therapeutic potential of KAS-08 for combination therapy in cancer treatment.

The Complexity of the cGAS-STING Pathway in CNS Pathologies.

Neuroinflammation driven by type-I interferons in the CNS is well established to exacerbate the progression of many CNS pathologies both acute and chronic. The role of adaptor protein Stimulator of Interferon Genes (STING) is increasingly appreciated to instigate type-I IFN-mediated neuroinflammation. As an upstream regulator of type-I IFNs, STING modulation presents a novel therapeutic opportunity to mediate inflammation in the CNS. This review will detail the current knowledge of protective and detrimental STING activity in acute and chronic CNS pathologies and the current therapeutic avenues being explored.

Abstract 3325: Intratumoral STING activation on artificially generated cutaneous metastasis of EML4-ALK lung cancer alters immunologic milieu of primary tumor

Abstract Introduction: Therapeutic monoclonal antibodies targeting immune checkpoints inhibitors (ICIs) are game changers in the treatment landscape of multiple solid tumors. However, response rate remains relatively low in most cases. Particularly, anti-PD-1 or PD-L1 therapies are not effective in EML4-ALK rearranged- and EGFR mutant-NSCLC patients due to their cold immune microenvironment. Recently, activation of stimulator of interferon genes (STING) has shown potential to enhance antitumor immunity through induction of various pro-inflammatory cytokines and chemokines. In this study, we attempted to induce inflamed tumor microenvironment by artificially generating skin metastasis using intratumoral STING activation in EML4-ALK transgenic mice. Methods: EML4-ALK transgenic mice were implanted with a new EML4-ALK tumor subcutaneously. The size of lung primary tumor and subcutaneous tumor were each measured by micro-MRI and calipers, respectively. The treatment groups were divided into the control group, tumor-implanted group, and intratumor STING activation group (skin). On day 14, subcutaneous tumor, primary tumor (lung), and systemic immune organs were collected for further analysis. Results: STING agonist promotes pIRF3 signaling and massive immune cell recruitment in skin region, the subcutaneous tumor was dramatically reduced with hyperinflammation. In addition, primary tumor (lung) was observed with increased T cell population in tumor with necrotic regions. IFN-gamma expression was increased in bronchoalveolar fluid in STING activation group compared to control and tumor only groups (P &amp;lt; 0.05). Tumor macrophages and NK cells were increased compared to control group (P &amp;lt; 0.05, P = ns, respectively). We considered that skin consists of cutaneous innate immune cells, especially, macrophage. Increased T cells and macrophages were observed in STING activated tumor within 6 hours. The in vitro assay proved that macrophages highly express chemokines (CCL5, CCL2, and CCL12) which is recruiting T cells and myeloid cells. This initial immune activation results in increased tumor-specific T cell, especially, memory helper T cell (CD3+CD4+CD44+) in spleen, LN and MLN. We tested anti-IFN-beta antibody for neutralization of STING activity in vivo. The IFN-gamma expression of lymphocytes in MLN was reduced but not significant. Conclusion: This study confirmed that STING activation in cold tumor alters inflamed-immunologic milieu of primary tumor via hyper cutaneous immune activity with pivotal roles of macrophage. Our findings provide a rationale for further clinical investigations. Keywords: STING, cutaneous metastasis, EML4-ALK Citation Format: Chun-Bong Synn, Ji Min Lee, SunMin Lim, Do Hee Kim, Bianca-Ioana Gheorghiu, Jae-Hwan Kim, Wongeun Lee, Sung-Eun Kim, Sungwon Park, Beung-Chul Ahn, Ha Ni Jo, Min Hee Hong, Hye Ryun Kim, Kyoung-Ho Pyo, Byung Chul Cho. Intratumoral STING activation on artificially generated cutaneous metastasis of EML4-ALK lung cancer alters immunologic milieu of primary tumor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3325.

The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT chemoradiation results in the death of dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) HSCT models. Here we show that STING rapidly promotes donor CD8⁺ T cell activation and recipient APC death early after aHSCT. Post MUD HSCT, we previously found a >2x reduction in IFNβ, TNFα and IL-6 mRNA in STING^−/− vs WT recipient colonic mRNA expression (48 hrs) as well as decreased weight loss, GVHD scores and skin pathology (6 wks) vs WT. Chimeric studies showed that STING loss in non-hematopoietic cells induced this effect. Conversely, we recently found a single early (<D14), but not late (>D100), dose of the STING agonist DMXAA increased GVHD scores and lethality in WT, but not STING^−/−, recipients. Thus, the activation of this pathway can promote GVHD following MUD HSCT. Furthermore, mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STING^HAQ/HAQ) also exhibited reduced GVHD after MUD HSCT, suggesting potential clinical importance. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD HSCT (Bader CS, et al, in revision <i>Sci. Transl. Med.</i>) contrasts reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) HSCT. Since CD4⁺ and CD8⁺ T cells are central in MMUD and MUD GVHD, respectively, we transplanted MMUD BALB/c BM + CD8⁺ T cells into B6-WT and STING^−/− mice and notably, found that STING^−/- recipients now developed reduced GVHD clinical scores, skin pathology and frequencies of activated T cells 8 wks post-HSCT vs WT. We also found that STING^−/− mice had greater numbers of recipient splenic CD11b⁺CD11c⁺ APCs and these cells expressed reduced MHC I protein 1 day after MMUD B6 into BALB/c aHSCT vs WT (Fig. A, B). Moreover, STING^−/− recipient spleens contained lower numbers of donor CD8⁺ T cells producing IFNγ and TNFα (Fig. C), supporting the hypothesis that STING contributes to early activation of donor CD8⁺ T cells and loss of recipient APCs. Next, to identify if reduced host MHC II⁺ APCs affected donor CD4⁺ T cell activation, B6-Nur77^GFP T cells were used to explicitly monitor T cell receptor signaling. Indeed, STING^−/− spleens had greater numbers of donor Nur77^GFP CD4⁺ T cells expressing GFP, CD69 and IFNγ 6 days post-HSCT (Fig. D). Overall, these data provide a mechanism by which STING could promote CD8⁺ T cell-mediated GVHD yet diminish CD4⁺-mediated GVHD. Ongoing studies are exploring the use of STING agonists to augment GVL following aHSCT.

The Innate Immune Sensor Sting Promotes Donor CD8+ T Cell Activation and Recipient APC Death Early after Preclinical Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplants (aHSCTs). Pre-HSCT chemoradiation results in the death of dividing cells and release of endogenous danger signals. These molecules drive the activation of antigen presenting cells (APCs) and the differentiation of allo-reactive donor T cells. We identified a role for Stimulator of Interferon Genes (STING), an innate immune sensor, in GVHD using pre-clinical MHC-matched unrelated donor (MUD) HSCT models. Here we show that STING rapidly promotes donor CD8+ T cell activation and recipient APC death early after aHSCT. Post MUD HSCT, we previously found a >2x reduction in IFNβ, TNFα and IL-6 mRNA in STING−/− vs WT recipient colonic mRNA expression (48 hrs) as well as decreased weight loss, GVHD scores and skin pathology (6 wks) vs WT. Chimeric studies showed that STING loss in non-hematopoietic cells induced this effect. Conversely, we recently found a single early ( D100), dose of the STING agonist DMXAA increased GVHD scores and lethality in WT, but not STING−/−, recipients. Thus, the activation of this pathway can promote GVHD following MUD HSCT. Furthermore, mice homozygous for a murine homologue of a human allele associated with diminished STING activity (STINGHAQ/HAQ) also exhibited reduced GVHD after MUD HSCT, suggesting potential clinical importance. Interestingly, our findings that STING deficiency ameliorates GVHD in MUD HSCT (Bader CS, et al, in revision Sci. Transl. Med.) contrasts reported observations that STING deficiency can exacerbate GVHD after MHC-mismatched (MMUD) HSCT. Since CD4+ and CD8+ T cells are central in MMUD and MUD GVHD, respectively, we transplanted MMUD BALB/c BM + CD8+ T cells into B6-WT and STING−/− mice and notably, found that STING−/- recipients now developed reduced GVHD clinical scores, skin pathology and frequencies of activated T cells 8 wks post-HSCT vs WT. We also found that STING−/− mice had greater numbers of recipient splenic CD11b+CD11c+ APCs and these cells expressed reduced MHC I protein 1 day after MMUD B6 into BALB/c aHSCT vs WT (Fig. A, B). Moreover, STING−/− recipient spleens contained lower numbers of donor CD8+ T cells producing IFNγ and TNFα (Fig. C), supporting the hypothesis that STING contributes to early activation of donor CD8+ T cells and loss of recipient APCs. Next, to identify if reduced host MHC II+ APCs affected donor CD4+ T cell activation, B6-Nur77GFP T cells were used to explicitly monitor T cell receptor signaling. Indeed, STING−/− spleens had greater numbers of donor Nur77GFP CD4+ T cells expressing GFP, CD69 and IFNγ 6 days post-HSCT (Fig. D). Overall, these data provide a mechanism by which STING could promote CD8+ T cell-mediated GVHD yet diminish CD4+-mediated GVHD. Ongoing studies are exploring the use of STING agonists to augment GVL following aHSCT.