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STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern asStat3deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediatedStat3deletion in 20% of the hematopoietic compartment.Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast toStat3-sufficient (CreER) controls. Single-cell RNA sequencing of Lin−ckit+Sca1+BM cells revealed altered transcriptional responses inStat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation,Stat3-deficient Lin−ckit+Sca1+cells accumulated γH2AX over time. Following secondary BM transplantation,Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs.Key PointsSTAT3 is critical for hematopoietic activity and hematopoietic stem cell maintenance in non-inflammatory conditionsSTAT3 has a cell-intrinsic role in the suppression of interferon signaling and myeloid-skewed transcription in hematopoietic stem cells

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.

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.

Introduction: Cryoablation is a non-surgical approach that uses rapid freeze-thaw cycles to kill cancer cells while preserving tumor-derived antigens. Our recent preclinical study in mice bearing bilateral high-risk metastatic breast tumors revealed cryoablation of the primary tumor enriched tumor-infiltrating lymphocytes in the non-treated abscopal tumor compared to resection, and prolonged overall mouse survival. Thus, it is clinically relevant to define the specific mechanisms by which cryoablation promotes antitumor immunity in abscopal tumors compared to resection. Here, we used RNA-sequencing (RNA-Seq) to identify immune gene signatures associated with enhanced abscopal effect following cryoablation versus resection in a murine model of metastatic breast cancer. Methods: BALB/c mice were implanted orthotopically and bilaterally with 4T1-12B (luciferase-expressing) cells. Two weeks after, left tumors were resected or cryoablated (tumor remained in mouse); right tumors were removed one week posttreatment to assess the abscopal immune response by RNA-Seq. Left tumors resected at two weeks were used as baseline controls. FASTQ files were trimmed and aligned to GRCm39; read counts were analyzed with edgeR and resulting differentially expressed genes with Ingenuity Pathway Analysis. Results: Cryoablation and resection uniquely affected the overall gene signatures and global activation of distinct signaling pathways in abscopal tumors compared to baseline and to each other. Since we observed differences in immune-related pathways, we further examined changes in the tumor immune landscape. Genes of various T cell populations and B cells were more upregulated in abscopal tumors following cryoablation compared to resection, suggesting cryoablation uniquely affects lymphocyte status. Within immune pathways in abscopal tumors following primary tumor cytoablation compared to resection, we found similar activation of Th1 signaling and greater activation of nitric oxide and reactive oxygen species production in macrophages, as well as Th2, NK cell, and IL-7 signaling. By contrast, crosstalk between dendritic cells and NK cells, PD-1/PD-L1 signaling, phagosome formation, and TREM1 signaling were less activated. Within these pathways, genes that promote angiogenesis and immunosuppression (Cxcl2) or metastasis (Cx3cr1) were downregulated in abscopal tumors following cryoablation compared to resection, whereas genes involved in T and NK cell-mediated cytolysis (perforin 1, Prf1), and tumor suppression (Stat6 and Nlrp12), were upregulated. Summary: We identified important pathways and genes involved in tumorigenesis and immune function, which may serve as biomarkers of the abscopal effect induced by cryoablation. Our findings suggest cryoablation reduces immunosuppression while enhancing T cell infiltration and cytotoxicity within abscopal tumors compared to resection. Future in silico and in vivo approaches will further define mechanisms by which cryoablation shapes the abscopal tumor immune landscape. Citation Format: Rachel L. Babcock, Dalia Martinez-Marin, Flavia Sardela de Miranda, Sonia Y. Khan, Maribel Castro, Isabel Castro-Piedras, Kevin Pruitt, Michael W. Melkus, Rakhshanda Layeequr Rahman. Identification of immune gene signatures as potential biomarkers of abscopal effect post-cryoablation of breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B09.

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.

Long-term survival of allo- and xenotransplanted immune-privileged Sertoli cells (SCs) is well documented suggesting that SCs can be used to deliver foreign proteins for cell-based gene therapy. The aim of this study was to use a lentivirus carrying proinsulin cDNA to achieve stable expression and lowering of blood glucose levels (BGLs). A SC line transduced with the lentivirus (MSC-LV-mI) maintained stable insulin expression in vitro. These MSC-LV-mI cells were transplanted and grafts were analyzed for cell survival, continued proinsulin mRNA, and insulin protein expression. All grafts contained MSC-LV-mI cells that expressed proinsulin mRNA and insulin protein. Transplantation of MSC-LV-mI cells into diabetic mice significantly lowered BGLs for 4 days after transplantation. Interestingly, in three transplanted SCID mice and one transplanted BALB/c mouse, the BGLs again significantly lowered by day 50 and 70, respectively. This is the first time SC transduced with a lentiviral vector was able to stably express insulin and lower BGLs. In conclusion, a SC line can be modified to stably express therapeutic proteins (e.g., insulin), thus taking us one step further in the use of SCs as an immune-privileged vehicle for cell-based gene therapy.

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.

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.

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.

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-&gamma, + 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.