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Type I conventional dendritic cells (cDC1s) are an essential Ag-presenting population required for generating adaptive immunity against intracellular pathogens and tumors. While the transcriptional control of cDC1 development is well understood, the mechanisms by which extracellular stimuli regulate cDC1 function remain unclear. We previously demonstrated that the cytokine-responsive transcriptional regulator STAT3 inhibits polyinosinic:polycytidylic acid [poly(I:C)]-induced cDC1 maturation and cDC1-mediated antitumor immunity in murine breast cancer, indicating an intrinsic, suppressive role for STAT3 in cDC1s. To probe transcriptional mechanisms regulating cDC1 function, we generated novel RNA sequencing datasets representing poly(I:C)-, IL-10–, and STAT3-mediated gene expression responses in murine cDC1s. Bioinformatics analyses indicated that poly(I:C) stimulates multiple inflammatory pathways independent of STAT3, while IL-10–activated STAT3 uniquely inhibits the poly(I:C)-induced type I IFN (IFN-I) transcriptional response. We validated this mechanism using purified cDC1s deficient for STAT3 or IFN signaling. Our data reveal IL-10–activated STAT3 suppresses production of IFN-β and IFN-γ, accrual of tyrosine phosphorylated STAT1, and IFN-stimulated gene expression in cDC1s after poly(I:C) exposure. Moreover, we found that maturation of cDC1s in response to poly(I:C) is dependent on the IFN-I receptor, but not the type II IFN receptor, or IFN-λ. Taken together, we elucidate an essential role for STAT3 in restraining autocrine IFN-I signaling in cDC1s elicited by poly(I:C) stimulation, and we provide novel RNA sequencing datasets that will aid in further delineating inflammatory and anti-inflammatory mechanisms in cDC1s.

Breast cancer is the most common female malignancy in both the developed and developing world. Doxorubicin is one of the most commonly used chemotherapies for breast cancer. Unfortunately, up to 60% of survivors report long-term chemotherapy-induced cognitive dysfunction (CICD) characterized by deficits in working memory, processing speed and executive function. Currently, no therapeutic standard for treating CICD exists. Here, we hypothesized that treatment with a blood-brain barrier permeable histone deacetylase 6 (HDAC6) inhibitor can successfully reverse long-term doxorubicin-induced cognitive dysfunction. Methods: The puzzle box test and novel object/place recognition test were used to assess cognitive function following a therapeutic doxorubicin dosing schedule in female mice. Mitochondrial function and morphology in neuronal synaptosomes were evaluated using the Seahorse XF24 extracellular flux analyzer and transmission electron microscopy, respectively. Hippocampal postsynaptic integrity was evaluated using immunofluorescence. Hippocampal microglia phenotype was determined using advanced imaging techniques and single-nucleus RNA sequencing. Results: A 14-day treatment with a blood-brain barrier permeable HDAC6 inhibitor successfully reversed long-term CICD in the domains of executive function, working and spatial memory. No significant changes in mitochondrial function or morphology in neuronal synaptosomes were detected. Long-term CICD was associated with a decreased expression of postsynaptic PSD95 in the hippocampus. These changes were associated with decreased microglial ramification and alterations in the microglia transcriptome that suggest a stage 1 disease-associated microglia (DAM) phenotype. HDAC6 inhibition completely reversed these doxorubicin-induced alterations, indicating a restoration of microglial homeostasis. Conclusion: Our results show that decreased postsynaptic integrity and a neurodegenerative microglia phenotype closely resembling stage 1 DAM microglia contribute to long-term CICD. Moreover, HDAC6 inhibition shows promise as an efficacious pharmaceutical intervention to alleviate CICD and improve quality of life of breast cancer survivors.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Background: Chronic inflammation, a hallmark of cancer, is associated with poor prognosis in human various malignancies. Predominantly, tumor associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs) are immune cells well characterized to promote inflammation, support tumor growth and metastasis, by secreting chemokines, serine proteases and reactive oxygen species. Increased TANs and MDSCs are predictive markers of both tumor progression and metastasis, suggesting that their inhibition may provide viable anti-tumor strategies. Neutrophil elastase (NE), a serine protease exclusively secreted by TANs and MDSCs, is a crucial mediator of inflammation and tumor progression. Recent preliminary studies from our group suggests that genetic deletion of Elane (encoding NE) inhibits lung metastasis in in vivo models of breast cancer. Yet the precise mechanism(s) by which NE promotes tumorigenesis and metastasis of breast cancer remains to be elucidated. To address this gap in knowledge, the current focus of our work is to identify the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis. Methods: To determine the prognostic significance of NE, we analyzed 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC) for infiltration of TANs. To examine the role of NE in breast cancer metastasis, we have generated Elane+/+ and Elane-/- mice in BALB/c and FVB/N genetic backgrounds, bearing 4T1 and PyMT tumors (orthotopic and spontaneous) respectively. Each of these mouse models develop lung metastasis in 80-90% in tumor-bearing mice within 1-3 months of primary tumor initiation. Results: Immunohistochemical staining of NE, a marker of TANs shows that higher infiltration of NE-positive TANs is associated with recurrence free survival with a hazard ratio of 3.4 (95% CI, 1.1-5.5). Genetic ablation of NE (Elane −/−) in the PyMT and 4T1 models strongly inhibits lung metastasis. Specifically, the number of lung foci was reduced by ~90% in PyMT Elane−/− mice compared to controls (21.8 vs. 2.7, respectively; p=0.0044). Similarly, ~50% decrease in the number of lung foci was observed in the 4T1 Elane−/− mice compared to control (7.8 vs. 3.4, respectively; p=0.0281). Comparison of inflammatory factors in the tumor microenvironment revealed that Csf3 (G-CSF), Cxcr2 (CXCR2) and Cxcl1 (CXCL1/KC) were significantly elevated in tumors from PyMT/4T1 Elane+/+ mice vs. PyMT/4T1 Elane-/- animals. Mechanistically, our result implies NE-dependent recruitment of immunosuppressive subsets (CXCL1 and related chemokines), thus sustaining inflammation at the primary tumor site. NE- dependent secretion of CXCL1 can further enrich for cancer stem-like cells (CSCs) via CXCR2 signaling, thus aiding metastasis. Lastly, we interrogated the efficacy reversible NE inhibitor AZD9668 in Elane+/+ mouse models. 100mg/kg daily treatment of AZD9668 reduced lung metastasis in PyMT mice by 94% compared to vehicle-treated mice (0.49+/- 0.21% vs. 0.03+/-0.01%; p=0.05). Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis in in vivo models of breast cancer. NE inhibitors thus far have only been clinically tested in COPD patients and their efficacy in breast cancer remains to be evaluated. Our preclinical studies presented here are likely to provide the much needed rationale for the use of this class of NE inhibitors as a viable treatment strategy for the metastatic breast cancer. Citation Format: Amriti Rajender Lulla, Said Akli, Taylor T Chrisikos, Marc A. Pina, Yifan Zhou, Haiyan Li, Stephanie S Watowich, Khandan Keyomarsi. Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-02.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producing cells that promote anti-viral immune responses and contribute to autoimmunity. Development of pDCs requires the transcriptional regulator E2-2 and is opposed by inhibitor of DNA binding 2 (Id2). Prior work indicates Id2 is induced in pDCs upon maturation and may affect pDC IFN-I production via suppression of E2-2, suggesting an important yet uncharacterized role in this lineage. We found TLR7 agonists stimulate Id2 mRNA and protein expression in pDCs. We further show that transcriptional activation of Id2 is dependent on the E2 ubiquitin-conjugating enzyme Ubc13, but independent of IFN-I signaling in response to TLR7 agonist stimulation. Nonetheless, conditional Id2 depletion in pDCs indicates Id2 is dispensable for TLR7 agonist-induced maturation and inhibition of E2-2 expression. Thus, we identify new mechanisms of Id2 regulation by Ubc13, which may be relevant for understanding Id2 gene regulation in other contexts, while ruling out major roles for Id2 in pDC responses to TLR7 agonists.

Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

Background Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103+ cDC1s in mice, CD141+ cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied. Methods We generated murine CD103+ cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+ cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+ cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+ cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+ cDC1s. Results In vitro-generated CD103+ cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+ cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+ cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ+ CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+ cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103+ cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. Conclusions Our data indicate an in vitro-generated CD103+ cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+ cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Conventional dendritic cells (cDCs) are a critical immune population, composed of multiple subsets, and responsible for controlling adaptive immunity and tolerance. Although migratory type 1 cDCs (CD103+ cDC1s in mice) are necessary to mount CD8+ T cell-mediated anti-tumor immunity, whether and how tumors modulate CD103+ cDC1 function remain understudied. Signal Transducer and Activator of Transcription 3 (STAT3) mediates the intracellular signaling of tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10; thus, we hypothesized that STAT3 restrained anti-tumor immune responses elicited by CD103+ cDC1s. Herein, we show that in vitro-derived STAT3-deficient (Stat3∆/∆) CD103+ cDC1s are refractory to the inhibitory effects of IL-10 on Toll-like receptor 3 (TLR3) agonist-induced maturation responses. In a tumor vaccination approach, we found Stat3∆/∆ CD103+ cDC1s restrained mammary gland tumor growth and increased mouse survival more effectively than STAT3-sufficient CD103+ cDC1s. In addition, vaccination with Stat3∆/∆ CD103+ cDC1s elicited increased amounts of tumor antigen-specific CD8+ T cells and IFN-γ+ CD4+ T cells in tumors and tumor-draining lymph nodes versus phosphate-buffered saline (PBS)-treated animals. Furthermore, IL-10 receptor-deficient CD103+ cDC1s controlled tumor growth to a similar degree as Stat3∆/∆ CD103+ cDC1s. Taken together, our data reveal an inhibitory role for STAT3 in CD103+ cDC1 maturation and regulation of anti-tumor immunity. Our results also suggest IL-10 is a key factor eliciting immunosuppressive STAT3 signaling in CD103+ cDC1s in breast cancer. Thus, inhibition of STAT3 in cDC1s may provide an important strategy to improve their efficacy in tumor vaccination approaches and cDC1-mediated control of anti-tumor immunity.

e14566 Background: Immune checkpoint blockade (ICB) therapy transformed clinical oncology by inducing durable responses and increasing survival rates in many types of cancers. However, ICB is effective in only in a subset of patients. Recent studies delineated the role of gut microbiome as both a biomarker and a therapeutic in ICB responsiveness. We aimed to increase understanding of the microbiome-immune system axis in ICB therapy by using antibiotics to knock out certain components of gut microbiome. Methods: We treated MC38 colon adenocarcinoma-bearing mice with a widely-used antibiotic, metronidazole, that is effective against anaerobic and protozoal infections including Clostridioides difficile – a major mediator of colitis. Metronidazole was administered via oral gavage or mixed in drinking water before and after tumor injections. Mice received twice weekly treatment with a-CTLA4 ICB. Results: Metronidazole treatment alone slowed the growth rate of MC38 tumors, consistent with the current literature regarding colon cancer murine models. When metronidazole treatment was combined with a-CTLA4 therapy, we found ̃90% complete tumor regression. In the metronidazole and a-CTLA4 combination group, we also observed an increase in the number of CD103+ type 1 conventional dendritic cells (cDC1s) in colon lamina propria, which suggests enhanced antigen sampling from lumen. Also, in the mesenteric lymph nodes (mLN), we detected upregulation of CD80 and CD86 co-stimulatory molecule expression on CX3CR1+ antigen presenting cells. Multiplex analysis of colon cytokines and colon pathology evaluation was comparable among groups, which implies a non-inflammatory environment in colon. Analysis of tumor-draining lymph nodes eight days after tumor injections showed higher expression of CD86 on CD103+ cDC1s implying superior anti-tumor immunity. 16S rRNA gene sequencing analysis of fecal samples revealed loss of Lachnospiraceae and enrichment of Bifidobacteriaceae and Sutterellaceae families in metronidazole-treated mice. Conclusions: Shifting microbiome composition with metronidazole treatment elicits a favorable anti-tumor immune response to a-CTLA4 treatment in murine colon adenocarcinoma.

Conventional dendritic cells (cDCs) are the primary antigen presenting cells of the immune system, and thus control adaptive immunity. Type 1 cDCs (cDC1s) are crucial for mounting protective T cell responses against tumors and viruses. Recently, we reported that interleukin (IL)-10 inhibits polyinosinic-polycytidylic acid (poly I:C; a toll-like receptor 3 agonist) induced maturation of cDC1s in a signal transducer and activator of transcription (STAT) 3-dependent manner. In addition, using a tumor vaccine approach, we demonstrated that STAT3-deficient (Stat3Δ/Δ) cDC1s are more capable of inducing anti-tumor immunity than STAT3-sufficient (Stat3fl/fl) cDC1s. To further investigate how STAT3 and IL-10 inhibit cDC1 function, we performed RNA-sequencing using Stat3Δ/Δ and Stat3fl/fl cDC1s treated with IL-10, poly I:C, or both IL-10 and poly I:C. Gene Set Enrichment Analysis revealed that although many inflammatory signaling pathways are enriched in cDC1s upon treatment with poly I:C, only the interferon-alpha (IFN-α) response pathway is inhibited by IL-10 and STAT3. Furthermore, Ingenuity Pathway Analysis identified IFNs, and components of IFN receptor signaling, such as STAT1, as the primary targets of IL-10/STAT3-mediated inhibition. Therefore, pathway analyses suggest IL-10 and STAT3 inhibit poly I:C-induced IFN signaling in cDC1s. Indeed, IL-10 was found to inhibit poly I:C-induced accumulation of phosphorylated STAT1 by immunoblot. Moreover, investigations into specific IFNs determined that IFN-β and IFN-γ, but not IFN-λ, induce IFN-stimulated genes in cDC1s. Currently, studies are underway to further dissect the role of IFNs in cDC1 function and IL-10/STAT3-mediated inhibition of poly I:C-induced maturation.

The inhibitor of DNA binding (Id) proteins and E proteins are basic helix-loop-helix proteins that act as mutual antagonists to direct immune cell development and function. In developing dendritic cells (DCs), Id2 specifies type 1 conventional DCs (cDC1s) and represses E2-2, an E protein and master regulator of plasmacytoid DCs (pDCs). DCs detect viruses, bacteria, and tumor cells via pattern recognition receptors, such as Toll-like receptors (TLRs), to elicit immune responses. Previously, we found Id2 mRNA and protein was induced in TLR agonist-stimulated murine pDCs. These data suggest TLR signaling alters the balance of Id and E proteins in DCs, however it is unknown whether TLR signaling affects Id2 mRNA in common DC progenitors (CDPs) and what effect this has on DC lineage development. We hypothesize TLR stimulation induces Id2 in DC progenitors thereby affecting generation of DC subsets. We found TLR agonist-stimulated CDPs expressed higher amounts of Id2 mRNA compared to non-treated controls. TLR ligand-treated mice exhibited a rapid decrease in CDPs that recovered within 24-hours, consistent with prior work, as well as changes in peripheral DC subset frequencies. Interestingly, Id2-deficient and -sufficient pDCs and cDC1s exhibited similar phenotypic and functional changes to TLR agonist stimulation, including co-stimulatory molecule expression and soluble factor production. These data suggest induction of Id2 occurs in CDPs in response to TLR stimulation and affects DC development, while Id2 appears to be dispensable for pDC and cDC1 maturation. Future work will test the requirement of Id2 in TLR agonist-induced DC lineage differentiation, and analyze the effect of cytokines on Id2 expression in DC progenitors.

Type 1 conventional dendritic cells (cDC1s) have critical roles in inducing adaptive immune responses and mediating immune tolerance. Signal Transducer and Activator of Transcription 5 (STAT5) was found to be crucial for monocyte-derived DC development; however, its role in non-lymphoid tissue CD103+cDC1s remained largely unknown. We evaluated the role of STAT5 by employing myeloid-specific Stat5-deficient mice,LysM-Cre +Stat5f/f and CD11c-Cre+Stat5f/f mice. Both strains show deficiencies in the numbers of CD103+cDC1s and alveolar macrophages (AMs) in lung, suggesting ineffective maintenance of lung homeostasis. Consistent with this concept, CD11c-Cre+Stat5f/f mice show granulocyte and monocyte accumulation in lung, accompanied by a reduction in the number of CD4+T, CD8+T, and B cells. Moreover, CD11c-Cre+Stat5-deficient mice have increased numbers of alveolar histiocytes, which are also enlarged, as well as accumulation of amorphous and eosinophilic material in alveolar spaces, alveolar histiocytosis, and proteinosis. In addition to the lung, liver CD103+cDC1s were also decreased by Stat5 depletion, however colon CD103+cDC1s were not affected. To examine cell-autonomous roles for STAT5, we generated Stat5-deficient CD103+cDC1s using our established bone marrow culture system, and tested in vitro-differentiated CD103+cDC1s for antigen presentation activity. These assays suggest Stat5 deficiency impaired CD103+cDC1s antigen cross-presentation ability. Taken together, our study showed that STAT5 is required for homeostasis and function of CD103+cDC1s in non-lymphoid organs.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producing cells that mediate anti-viral responses, anti-tumor immunity, and autoimmunity. When exposed to Toll-like receptor 7 (TLR7)- and TLR9-ligands, pDCs mature and produce IFN-Is; pDCs also acquire conventional DC (cDC)-like morphology and features, including the cell surface expression of antigen presentation and co-stimulatory molecules, secretion of additional cytokines, and ability to activate adaptive T cell responses. Yet, the transcriptional pathways regulating TLR-induced pDC maturation are poorly characterized. We found the mRNA and protein expression of the inhibitor of DNA binding protein 2 (Id2) was induced in TLR7/9-stimulated pDCs, and Id2 expression correlated with greater cDC-like features. Id2 normally blocks pDC development by antagonizing the pDC-master regulator, E2-2, while E2-2 suppresses Id2 transcription. Thus, we hypothesize TLR-activated signaling pathways in pDCs control the abundance of transcriptional regulators (e.g., E2-2) at the Id2 promoter as well as the promoter chromatin state, leading to induction of Id2 transcription during pDC maturation. Using genetic knockout mouse models, we found Id2 induction was independent of interferon alpha receptor and nuclear factor kappa beta signaling pathways. Chromatin-immunoprecipitation assays suggested E2-2 abundance at the Id2 promoter decreased following TLR stimulation, while activating histone modifications increased. These data are consistent with transcriptional induction of Id2 upon TLR stimulation. Future work will investigate the effect of additional regulators and TLR-regulated pathways on Id2 induction in TLR7/9-stimulated pDCs.

Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, yet the efficacy of immunotherapy utilizingthis population is understudied. We used in vitro-generated CD103+cDC1s in vaccination strategies to test their ability to control melanoma and osteosarcoma tumors. In vitro-generated CD103+cDC1s produced cDC1-associated factors such as IL-12p70 and CXCL10, and demonstrated antigen cross-presentation activity upon stimulation with the Toll-like receptor 3 (TLR3) agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+cDC1s also migrated to tumor-draining lymph nodes (TdLNs) following poly I:C treatment and intratumoral (i.t.) delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+cDC1s enhanced tumor infiltration of tumor antigen-specific and IFN-g+CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+cDC1s showed superior anti-tumor efficacy compared to vaccination with monocyte-derived DCs (MoDCs), and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In addition, CD103+cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. Our data indicate an in vitro-generated CD103+cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Intestinal infections trigger immune responses that in some cases become dysregulated and lead to acute or chronic disease; understanding immune regulatory mechanisms in infection will help advance new therapies for intestinal disorders. Citrobacter rodentium infection in mice mimics enteropathogenic Escherichia coli infections in humans. We found that Leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is upregulated in colon and serum following C. rodentium infection; however, the source, targets and effects of this cytokine during intestinal disease remain unclear. To investigate the role of LIF during infection, we generated mice with LIF receptor (LIFR) deficiency in endothelial and hematopoietic cells (Tie2cre Lifrf/f) or CD11c+ cells (CD11c cre Lifrf/f). Upon C. rodentium infection, Tie2cre Lifrf/fandCD11c cre Lifrf/f mice had reduced colon length, increased bacterial dissemination to spleen and liver, and greater colon pathology scores relative to infected controls. Comparison of cytokine and chemokine expression in the colon revealed upregulation of various factors in infected LIFR-deficient mice, with the pro-inflammatory cytokine IL-23 significantly increased in CD11c cre Lifrf/f mice. Our preliminary data from LIFR-deficient, bone marrow-derived myeloid cells suggests LIFR signaling inhibits inflammatory cytokine production in vitro. Collectively, our results suggest LIFR signaling in CD11c+ cells inhibits colonic IL-23 production and protects from immune dysregulation during C. rodentium infection. Our current studies are investigating LIF-producing and -responding populations in colon, to elucidate protective immune mechanisms during infection.

Conventional dendritic cells (cDCs) are a crucial immune population, which includes multiple subtypes that coordinate adaptive immunity and tolerance. Migratory type 1 cDC1s (CD103+ cDC1s in mice) are required to induce CD8+ T cell-mediated anti-tumor immunity, yet whether and how tumors regulate CD103+ cDC1 function is largely unknown. Many tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10, require Signal Transducer and Activator of Transcription 3 (STAT3) for intracellular signaling; therefore, we sought to determine whether STAT3 inhibits CD103+ cDC1-mediated anti-tumor immunity. Indeed, we found that in vitro-derived STAT3-deficient (Stat3Δ/Δ) CD103+ cDC1sare resistant to IL-10 – mediated inhibition of Toll-like receptor 3 (TLR3)-agonist induced maturation. When used as an anti-tumor vaccine, Stat3Δ/Δ CD103+ cDC1s inhibited breast tumor growth and increased mouse survival to a greater degree than STAT3-sufficient CD103+ cDC1s. Vaccination with Stat3Δ/Δ CD103+ cDC1s increased tumor antigen-specific CD8+ T cells and IFN-g+ CD4+ T cells in tumors and tumor-draining lymph nodes compared to controls. Moreover, IL-10 receptor-deficient CD103+ cDC1s restrained tumor growth as effectively as Stat3Δ/Δ CD103+ cDC1s. Taken together, our results demonstrate STAT3 suppresses CD103+ cDC1 maturation and vaccine efficacy in breast cancer. The data also suggest IL-10 is an important factor in the murine mammary tumor environment mediating immunosuppressive STAT3 signaling in CD103+ cDC1s. Thus, STAT3 inhibition may provide an effective strategy for improving the anti-tumor efficacy of CD103+ cDC1-based vaccines.

BackgroundType 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103+cDC1s in mice, CD141+cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied.MethodsWe generated murine CD103+cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+cDC1s.ResultsIn vitro-generated CD103+cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ+CD8+T cells, and suppressed melanoma and osteosarcoma growth. CD103+cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103+cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases.ConclusionsOur data indicate an in vitro-generated CD103+cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage− Sca-1+ c-Kit+ CD150+ CD48− HSPC subset (LSK CD150+ CD48− cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48− cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.

Dendritic cells (DCs) are the principal antigen-presenting cells of the immune system and play key roles in controlling immune tolerance and activation. As such, DCs are chief mediators of tumor immunity. DCs can regulate tolerogenic immune responses that facilitate unchecked tumor growth. Importantly, however, DCs also mediate immune-stimulatory activity that restrains tumor progression. For instance, emerging evidence indicates the cDC1 subset has important functions in delivering tumor antigens to lymph nodes and inducing antigen-specific lymphocyte responses to tumors. Moreover, DCs control specific therapeutic responses in cancer including those resulting from immune checkpoint blockade. DC generation and function is influenced profoundly by cytokines, as well as their intracellular signaling proteins including STAT transcription factors. Regardless, our understanding of DC regulation in the cytokine-rich tumor microenvironment is still developing and must be better defined to advance cancer treatment. Here, we review literature focused on the molecular control of DCs, with a particular emphasis on cytokine- and STAT-mediated DC regulation. In addition, we highlight recent studies that delineate the importance of DCs in anti-tumor immunity and immune therapy, with the overall goal of improving knowledge of tumor-associated factors and intrinsic DC signaling cascades that influence DC function in cancer.