Summary To maintain a symbiotic relationship between the host and its resident intestinal microbiota, appropriate mucosal T cell responses to commensal antigens must be established. Mice acquire both IgG and IgA maternally; the former has primarily been implicated in passive immunity to pathogens while the latter mediates host-commensal mutualism. Here we report the surprising observation that mice generate T cell independent and largely Toll-like receptor (TLR) dependent IgG2b and IgG3 antibody responses against their gut microbiota. We demonstrate that maternal acquisition of these antibodies dampens mucosal T follicular helper responses and subsequent germinal center B cell responses following birth. This work reveals a feedback loop whereby T cell independent, TLR-dependent antibodies limit mucosal adaptive immune responses to newly acquired commensal antigens and uncovers a broader function for maternal IgG.

Intratumoral (IT) STING activation results in tumor regression in preclinical models, yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here, clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to highdose tumor ablative regimens that result in systemic S100 distribution, low-dose immunogenic regimens induce local activation of tumor-specific CD8 + effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation, and in the context of optimized T cell responses, TNFa is dispensable for tumor control. In a poorly immunogenic model, S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity.

In the originally published version of this article, the 4T1 tumor cell line described in Figures 1, 2, and 5 and Supplemental Figures S1, S2, and S5 was mis-identified. This cell line has since been identified via short tandem repeat analysis as the murine CT26 tumor cell line, which is an undifferentiated colon carcinoma cell line. Furthermore, the line that was used had been engineered to express the human mesothelin protein. The authors believe that the substance and interpretation of the experiments put forth in the article remain the same. The online article, Figures 2 and S1, and the Supplemental Information have been updated to reflect the correction.

The cGAS-STING cytosolic DNA sensing pathway may play an integral role in the initiation of antitumor immune responses. Studies evaluating the immunogenicity of various cyclic dinucleotide (CDN) STING agonists administered by intratumoral (i.t.) injection showed potent induction of inflammation, tumor necrosis, and, in some cases, durable tumor-specific adaptive immunity. However, the specific immune mechanisms underlying these responses remain incompletely defined. The majority of these studies have focused on the effect of CDNs on immune cells but have not conclusively interrogated the role of stromal cells in the acute rejection of the CDN-injected tumor. Here, we revealed a mechanism of STING agonist-mediated tumor response that relied on both stromal and immune cells to achieve tumor regression and clearance. Using knockout and bone marrow chimeric mice, we showed that although bone marrow–derived TNFα was necessary for CDN-induced necrosis, STING signaling in radioresistant stromal cells was also essential for CDN-mediated tumor rejection. These results provide evidence for crosstalk between stromal and hematopoietic cells during CDN-mediated tumor collapse after i.t. administration. These mechanistic insights may prove critical in the clinical development of STING agonists.

Summary Pathogens utilize features of the host response as cues to regulate virulence gene expression. Salmonella enterica serovar Typhimurium (ST) sense Toll-like receptor (TLR)-dependent signals to induce Salmonella Pathogenicity Island 2 (SPI2), a locus required for intracellular replication. To examine pathogenicity in the absence of such cues, we evaluated ST virulence in mice lacking all TLR function (Tlr2−/−×Tlr4−/−×Unc93b13d/3d). When delivered systemically to TLR-deficient mice, ST do not require SPI2 and maintain virulence by replicating extracellularly. In contrast, SPI2 mutant ST are highly attenuated after oral infection of the same mice, revealing a role for SPI2 in the earliest stages of infection, even when intracellular replication is not required. This early requirement for SPI2 is abolished in MyD88−/− xTRIF−/− mice lacking both TLR- and other MyD88-dependent signaling pathways, a potential consequence of compromised intestinal permeability. These results demonstrate how pathogens use plasticity in virulence strategies to respond to different host immune environments.

Endothelial dysfunction and vascular leak, pathogenic hallmarks of severe dengue disease, are directly triggered by dengue virus (DENV) nonstructural protein 1 (NS1). Previous studies have shown that immunization with NS1, as well as passive transfer of NS1-immune serum or anti-NS1 mAb, prevent NS1-mediated lethality in vivo. In this study, we evaluated the immunogenicity and protective capacity of recombinant DENV NS1 administered with cyclic dinucleotides (CDNs), potent activators of innate immune pathways and highly immunogenic adjuvants. Using both wild-type C57BL/6 mice and IFN-a/b receptor-deficient mice, we show that NS1-CDN immunizations elicit serotype-specific and cross-reactive Ab and T cell responses. Furthermore, NS1-CDN vaccinations conferred significant homotypic and heterotypic protection from DENV2-induced morbidity and mortality. In addition, we demonstrate that high anti-NS1 Ab titers are associated with protection, supporting the role of humoral responses against DENV NS1 as correlates of protection. These findings highlight the potential of CDN-based adjuvants for inducing Ab and T cell responses and validate NS1 as an important candidate for dengue vaccine development.

Innate immune sensing of tumors is a critical step in generating spontaneous anti-tumor T cell responses. Endogenous activation of the STING pathway in immune cells and the subsequent generation of type I IFN is sufficient to generate spontaneous anti-tumor T cell responses. To take advantage of this tumor defense mechanism for therapeutic intervention, Aduro has developed the first-in-class STING agonist, ADU-S100 (MIW815), a small molecule derivative of the natural cyclic dinucleotide STING ligand. In mouse models, intratumoral administration of ADU-S100(MIW815) increases systemic tumor-specific T cells and results in substantial antitumor efficacy. ADU-S100 (MIW815) is currently being tested as mono- or combination therapy in Phase 1 clinical studies enrolling patients with cutaneously-accessible treatment-refractory advanced solid tumors and lymphomas. STING is broadly expressed across different cell types, however several studies have demonstrated that tumor cells regulate expression of STING and other members of the pathway, mainly cGAS, by epigenetic mechanisms. Activation of the STING pathway in innate immune cells is necessary for the generation of anti-tumor T cell responses, but the role of other cell types within the tumor microenvironment in response or resistance to STING agonists is not completely understood. To understand the contribution of tumor-cell STING to the anti-tumor response, we generated STING-deficient 4T1 tumor cells using the CRISPR/Cas9 system. Composition of the tumor microenvironment, endogenous T cell responses and tumor growth were comparable in animals with implanted STING-WT or -KO tumor cells. Similar data was observed in the B16.SIY melanoma model. In order to understand the role of tumor-STING in the context of ADU-S100 (MIW815) treatment, animals implanted with 4T1 STING WT or KO tumor cells were treated with a wide range of ADU-S100 doses. Expression of STING within tumor cells did not impact activation of innate cells or generation of tumor-specific T cells among all the tested doses. At immunogenic doses, tumor cell expression of STING was not required for tumor growth control.Overall, these results show that ADU-S100 (MIW815) activation of STING in host cells rather than in tumor cells is critical for production of type I interferon and tumor control. This supports potential treatment of cancers of different histologies regardless of tumor cell-intrinsic STING expression. Citation Format: Leticia Corrales, Antony L. Desbien, Kelsey E. Sivick Gauthier, Weiwen Deng, Tamara Schroeder, Gabrielle L. Reiner, Natalie Surh, Brian Francica, Ken Metchette, Chudi O. Ndubaku, Jeffrey M. McKenna, Yan Feng, Lianxing Zheng, Steven L. Bender, Charles Y. Cho, Andrea van Elsas, Meredith L. Leong, Sarah M. McWhirter. Tumor cell intrinsic STING signaling demonstrates minimal contribution to the anti-tumor response elicited by the STING agonist ADU-S100 (MIW815) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1202.

Activation of the STING pathway by intratumoral (IT) injection of synthetic cyclic dinucleotides (CDNs) induces stable tumor regression in preclinical models, yet the underlying immune correlates are not fully understood. ADU-S100, a CDN under clinical evaluation, was administered IT with an optimized dosing regimen to explore the immune requirements for antitumor efficacy in mouse syngeneic tumor models. We show that CD8+ T cells are necessary and sufficient for durable antitumor immunity elicited by ADU-S100 and that activation of STING in hematopoietic cells mediates CD8+ T cell induction. Both type I IFN and TNFα, which are induced by STING pathway activation, influence the antitumor immune response. The combination of ADU-S100 and anti-PD1 treatment enhances CD8+ T cell-dependent, noninjected tumor control that correlates with an enhanced effector profile of CD8+ T cells in the tumor. Combination of ADU-S100 with checkpoint inhibition also enhances durable immunity in a poorly immunogenic tumor model. Together, these results elucidate the immune correlates to STING-mediated antitumor efficacy and highlight the potential of combining STING agonists with checkpoint inhibition in the clinic. Citation Format: Anthony L. Desbien, Kelsey Sivick Gauthier, Leticia Corrales, Gabrielle Reiner, Laura Hix Glickman, George Katibah, Thomas E. Hudson, Uyen Vu, Natalie H. Surh, Brian Francica, Weiwen Deng, David B. Kanne, Justin J. Leong, Chudi Ndubaku, Ken Metchette, Jeffery M. McKenna, Steven L. Bender, Meredith L. Leong, Thomas W. Dubensky Jr., Andrea van Elsas, Sarah M. McWhirter. Intratumoral activation of STING with a synthetic cyclic dinucleotide elicits antitumor CD8 T-cell immunity that effectively combines with checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 631.