Intestinal microbiota signatures of clinical response and immune-related adverse events in melanoma patients treated with anti-PD-1.

Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with antitumor response to anti-programmed cell death protein-1 (PD-1) therapy, but inconsistencies exist between published microbial signatures associated with clinical outcomes. To resolve this, we evaluated a new melanoma cohort, along with four published datasets. Time-to-event analysis showed that baseline microbiota composition was optimally associated with clinical outcome at approximately 1 year after initiation of treatment. Meta-analysis and other bioinformatic analyses of the combined data show that bacteria associated with favorable response are confined within the Actinobacteria phylum and the Lachnospiraceae/Ruminococcaceae families of Firmicutes. Conversely, Gram-negative bacteria were associated with an inflammatory host intestinal gene signature, increased blood neutrophil-to-lymphocyte ratio, and unfavorable outcome. Two microbial signatures, enriched for Lachnospiraceae spp. and Streptococcaceae spp., were associated with favorable and unfavorable clinical response, respectively, and with distinct immune-related adverse effects. Despite between-cohort heterogeneity, optimized all-minus-one supervised learning algorithms trained on batch-corrected microbiome data consistently predicted outcomes to programmed cell death protein-1 therapy in all cohorts. Gut microbial communities (microbiotypes) with nonuniform geographical distribution were associated with favorable and unfavorable outcomes, contributing to discrepancies between cohorts. Our findings shed new light on the complex interaction between the gut microbiome and response to cancer immunotherapy, providing a roadmap for future studies.

Expression of Nutrient Transporters on NK Cells During Murine Cytomegalovirus Infection Is MyD88-Dependent.

Natural killer (NK) cells are the predominant innate lymphocytes that provide early defense against infections. In the inflammatory milieu, NK cells modify their metabolism to support high energy demands required for their proliferation, activation, and functional plasticity. This metabolic reprogramming is usually accompanied by the upregulation of nutrient transporter expression on the cell surface, leading to increased nutrient uptake required for intense proliferation. The interleukin-1 family members of inflammatory cytokines are critical in activating NK cells during infection; however, their underlying mechanism in NK cell metabolism is not fully elucidated. Previously, we have shown that IL-18 upregulates the expression of solute carrier transmembrane proteins and thereby induces a robust metabolic boost in NK cells. Unexpectedly, we found that IL-18 signaling is dispensable during viral infection in vivo, while the upregulation of nutrient transporters is primarily MyD88-dependent. NK cells from Myd88-/- mice displayed significantly reduced surface expression of nutrient receptors and mTOR activity during MCMV infection. We also identified that IL-33, another cytokine employing MyD88 signaling, induces the expression of nutrient transporters but requires a pre-exposure to IL-12. Moreover, signaling through the NK cell activating receptor, Ly49H, can also promote the expression of nutrient transporters. Collectively, our findings revealed multiple pathways that can induce the expression of nutrient transporters on NK cells while highlighting the imperative role of MyD88 in NK cell metabolism during infection.

Tristetraprolin expression by keratinocytes protects against skin carcinogenesis.

Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis, or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA-binding proteins to AU-rich elements (AREs) located in their 3'-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP; encoded by Zfp36), is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of cutaneous chemical carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controlled both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes and show that EGFR signaling potentially contributed to exacerbated tumor formation. Finally, single-cell RNA-Seq analysis indicated that ZFP36 was downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells played a major role in the control of skin tumorigenesis.

Human NK cells prime inflammatory DC precursors to induce Tc17 differentiation.

Adaptive immune responses are acknowledged to evolve from innate immunity. However, limited information exists regarding whether encounters between innate cells direct the generation of specialized T-cell subsets. We aim to understand how natural killer (NK) cells modulate cell-mediated immunity in humans. We found that human CD14+CD16- monocytes that differentiate into inflammatory dendritic cells (DCs) are shaped at the early stages of differentiation by cell-to-cell interactions with NK cells. Although a fraction of monocytes is eliminated by NK-cell-mediated cytotoxicity, the polarization of interferon-γ (IFN-γ) at the NKp30-stabilized synapses triggers a stable IFN-γ signature in surviving monocytes that persists after their differentiation into DCs. Notably, NK-cell-instructed DCs drive the priming of type 17 CD8+ T cells (Tc17) with the capacity to produce IFN-γ and interleukin-17A. Compared with healthy donors, this cellular network is impaired in patients with classical NK-cell deficiency driven by mutations in the GATA2 gene. Our findings reveal a previously unrecognized connection by which Tc17-mediated immunity might be regulated by NK-cell-mediated tuning of antigen-presenting cells.

Interleukin-18 and cytotoxic impairment are independent and synergistic causes of murine virus-induced hyperinflammation.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes typically associated with underlying hematologic and rheumatic diseases, respectively. Familial HLH is associated with genetic cytotoxic impairment and thereby to excessive antigen presentation. Extreme elevation of serum interleukin-18 (IL-18) has been observed specifically in patients with MAS, making it a promising therapeutic target, but how IL-18 promotes hyperinflammation remains unknown. In an adjuvant-induced MAS model, excess IL-18 promoted immunopathology, whereas perforin deficiency had no effect. To determine the effects of excess IL-18 on virus-induced immunopathology, we infected Il18-transgenic (Il18tg) mice with lymphocytic choriomeningitis virus (LCMV; strain Armstrong). LCMV infection is self-limited in wild-type mice, but Prf1-/- mice develop prolonged viremia and fatal HLH. LCMV-infected Il18-transgenic (Il18tg) mice developed cachexia and hyperinflammation comparable to Prf1-/- mice, albeit with minimal mortality. Like Prf1-/- mice, immunopathology was largely rescued by CD8 depletion or interferon-γ (IFNg) blockade. Unlike Prf1-/- mice, they showed normal target cell killing and normal clearance of viral RNA and antigens. Rather than impairing cytotoxicity, excess IL-18 acted on T lymphocytes to amplify their inflammatory responses. Surprisingly, combined perforin deficiency and transgenic IL-18 production caused spontaneous hyperinflammation specifically characterized by CD8 T-cell expansion and improved by IFNg blockade. Even Il18tg;Prf1-haplosufficient mice demonstrated hyperinflammatory features. Thus, excess IL-18 promotes hyperinflammation via an autoinflammatory mechanism distinct from, and synergistic with, cytotoxic impairment. These data establish IL-18 as a potent, independent, and modifiable driver of life-threatening innate and adaptive hyperinflammation and support the rationale for an IL-18-driven subclass of hyperinflammation.

T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis.

Cancer development requires a favorable tissue microenvironment. By deleting Myd88 in keratinocytes or specific bone marrow subpopulations in oncogenic RAS-mediated skin carcinogenesis, we show that IL17 from infiltrating T cells and IκBζ signaling in keratinocytes are essential to produce a permissive microenvironment and tumor formation. Both normal and RAS-transformed keratinocytes respond to tumor promoters by activating canonical NF-κB and IκBζ signaling, releasing specific cytokines and chemokines that attract Th17 cells through MyD88-dependent signaling in T cells. The release of IL17 into the microenvironment elevates IκBζ in normal and RAS-transformed keratinocytes. Activation of IκBζ signaling is required for the expression of specific promoting factors induced by IL17 in normal keratinocytes and constitutively expressed in RAS-initiated keratinocytes. Deletion of Nfkbiz in keratinocytes impairs RAS-mediated benign tumor formation. Transcriptional profiling and gene set enrichment analysis of IκBζ-deficient RAS-initiated keratinocytes indicate that IκBζ signaling is common for RAS transformation of multiple epithelial cancers. Probing The Cancer Genome Atlas datasets using this transcriptional profile indicates that reduction of IκBζ signaling during cancer progression associates with poor prognosis in RAS-driven human cancers. IMPLICATIONS: The paradox that elevation of IκBζ and stimulation of IκBζ signaling through tumor extrinsic factors is required for RAS-mediated benign tumor formation while relative IκBζ expression is reduced in advanced cancers with poor prognosis implies that tumor cells switch from microenvironmental dependency early in carcinogenesis to cell-autonomous pathways during cancer progression.

Commensal-specific T cell plasticity promotes rapid tissue adaptation to injury.

Barrier tissues are primary targets of environmental stressors and are home to the largest number of antigen-experienced lymphocytes in the body, including commensal-specific T cells. We found that skin-resident commensal-specific T cells harbor a paradoxical program characterized by a type 17 program associated with a poised type 2 state. Thus, in the context of injury and exposure to inflammatory mediators such as interleukin-18, these cells rapidly release type 2 cytokines, thereby acquiring contextual functions. Such acquisition of a type 2 effector program promotes tissue repair. Aberrant type 2 responses can also be unleashed in the context of local defects in immunoregulation. Thus, commensal-specific T cells co-opt tissue residency and cell-intrinsic flexibility as a means to promote both local immunity and tissue adaptation to injury.

Microbes and Cancer.

Commensal microorganisms (the microbiota) live on all the surface barriers of our body and are particularly abundant and diverse in the distal gut. The microbiota and its larger host represent a metaorganism in which the cross talk between microbes and host cells is necessary for health, survival, and regulation of physiological functions locally, at the barrier level, and systemically. The ancestral molecular and cellular mechanisms stemming from the earliest interactions between prokaryotes and eukaryotes have evolved to mediate microbe-dependent host physiology and tissue homeostasis, including innate and adaptive resistance to infections and tissue repair. Mostly because of its effects on metabolism, cellular proliferation, inflammation, and immunity, the microbiota regulates cancer at the level of predisposing conditions, initiation, genetic instability, susceptibility to host immune response, progression, comorbidity, and response to therapy. Here, we review the mechanisms underlying the interaction of the microbiota with cancer and the evidence suggesting that the microbiota could be targeted to improve therapy while attenuating adverse reactions.

Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment.

The gut microbiota influences both local and systemic inflammation. Inflammation contributes to development, progression, and treatment of cancer, but it remains unclear whether commensal bacteria affect inflammation in the sterile tumor microenvironment. Here, we show that disruption of the microbiota impairs the response of subcutaneous tumors to CpG-oligonucleotide immunotherapy and platinum chemotherapy. In antibiotics-treated or germ-free mice, tumor-infiltrating myeloid-derived cells responded poorly to therapy, resulting in lower cytokine production and tumor necrosis after CpG-oligonucleotide treatment and deficient production of reactive oxygen species and cytotoxicity after chemotherapy. Thus, optimal responses to cancer therapy require an intact commensal microbiota that mediates its effects by modulating myeloid-derived cell functions in the tumor microenvironment. These findings underscore the importance of the microbiota in the outcome of disease treatment.

MyD88 and its divergent toll in carcinogenesis.

Toll-like and interleukin-1 (IL-1) family receptors recognize microbial or endogenous ligands and inflammatory mediators, respectively, and with the exception of Toll-like receptor 3 (TLR3), signal via the adaptor molecule myeloid differentiation factor 88 (MyD88). MyD88 is involved in oncogene-induced cell intrinsic inflammation and in cancer-associated extrinsic inflammation, and as such MyD88 contributes to skin, liver, pancreatic, and colon carcinogenesis, as well as sarcomagenesis. MyD88 is also protective, for example in oncogenic virus carcinogenesis or, acting downstream of IL-18R to strengthen mucosal repair, in azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon carcinogenesis. Here, we discuss the mechanisms of the divergent effects of MyD88 and the balance of its protumor role in cancer-enhancing inflammation and immunity and its antitumor role in tissue homeostasis, repair, and immunity against the tumor or oncogenic pathogens.

Laser capture microdissection for protein and NanoString RNA analysis.

Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus(XT) instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.

Resurrection of endogenous retroviruses in antibody-deficient mice.

The mammalian host has developed a long-standing symbiotic relationship with a considerable number of microbial species. These include the microbiota on environmental surfaces, such as the respiratory and gastrointestinal tracts, and also endogenous retroviruses (ERVs), comprising a substantial fraction of the mammalian genome. The long-term consequences for the host of interactions with these microbial species can range from mutualism to parasitism and are not always completely understood. The potential effect of one microbial symbiont on another is even less clear. Here we study the control of ERVs in the commonly used C57BL/6 (B6) mouse strain, which lacks endogenous murine leukaemia viruses (MLVs) able to replicate in murine cells. We demonstrate the spontaneous emergence of fully infectious ecotropic MLV in B6 mice with a range of distinct immune deficiencies affecting antibody production. These recombinant retroviruses establish infection of immunodeficient mouse colonies, and ultimately result in retrovirus-induced lymphomas. Notably, ERV activation in immunodeficient mice is prevented in husbandry conditions associated with reduced or absent intestinal microbiota. Our results shed light onto a previously unappreciated role for immunity in the control of ERVs and provide a potential mechanistic link between immune activation by microbial triggers and a range of pathologies associated with ERVs, including cancer.

IL-1R-MyD88 signaling in keratinocyte transformation and carcinogenesis.

Constitutively active RAS plays a central role in the development of human cancer and is sufficient to induce tumors in two-stage skin carcinogenesis. RAS-mediated tumor formation is commonly associated with up-regulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. In this study, we report that mice lacking IL-1R or MyD88 are less sensitive to topical skin carcinogenesis than their respective wild-type (WT) controls. MyD88(-/-) or IL-1R(-/-) keratinocytes expressing oncogenic RAS are hyperproliferative and fail to up-regulate proinflammatory genes or down-regulate differentiation markers characteristic of RAS-expressing WT keratinocytes. Although RAS-expressing MyD88(-/-) keratinocytes form only a few small tumors in orthotopic grafts, IL-1R-deficient RAS-expressing keratinocytes retain the ability to form tumors in orthotopic grafts. Using both genetic and pharmacological approaches, we find that the differentiation and proinflammatory effects of oncogenic RAS in keratinocytes require the establishment of an autocrine loop through IL-1α, IL-1R, and MyD88 leading to phosphorylation of IκBα and NF-κB activation. Blocking IL-1α-mediated NF-κB activation in RAS-expressing WT keratinocytes reverses the differentiation defect and inhibits proinflammatory gene expression. Collectively, these results demonstrate that MyD88 exerts a cell-intrinsic function in RAS-mediated transformation of keratinocytes.

Compartmentalized control of skin immunity by resident commensals.

Intestinal commensal bacteria induce protective and regulatory responses that maintain host-microbial mutualism. However, the contribution of tissue-resident commensals to immunity and inflammation at other barrier sites has not been addressed. We found that in mice, the skin microbiota have an autonomous role in controlling the local inflammatory milieu and tuning resident T lymphocyte function. Protective immunity to a cutaneous pathogen was found to be critically dependent on the skin microbiota but not the gut microbiota. Furthermore, skin commensals tuned the function of local T cells in a manner dependent on signaling downstream of the interleukin-1 receptor. These findings underscore the importance of the microbiota as a distinctive feature of tissue compartmentalization, and provide insight into mechanisms of immune system regulation by resident commensal niches in health and disease.

The proinflammatory myeloid cell receptor TREM-1 controls Kupffer cell activation and development of hepatocellular carcinoma.

Chronic inflammation drives liver cancer pathogenesis, invasion, and metastasis. Liver Kupffer cells have crucial roles in mediating the inflammatory processes that promote liver cancer, but the mechanistic basis for their contributions are not fully understood. Here we show that expression of the proinflammatory myeloid cell surface receptor TREM-1 expressed by Kupffer cells is a crucial factor in the development and progression of liver cancer. Deletion of the murine homolog Trem1 in mice attenuated hepatocellular carcinogenesis triggered by diethylnitrosamine (DEN). Trem1 deficiency attenuated Kupffer cell activation by downregulating transcription and protein expression of interleukin (IL)-6, IL-1ß, TNF, CCL2, and CXCL10. In addition, Trem1 ablation diminished activation of the p38, extracellular regulated kinase 1/2, JNK, mitogen-activated protein kinase, and NF-κB signaling pathways in Kupffer cells, resulting in diminished liver injury after DEN exposure. Adoptive transfer of wild-type Kupffer cells to Trem1-deficient mice complemented these defects and reversed unresponsiveness to DEN-induced liver injury and malignant development. Together, our findings offer causal evidence that TREM-1 is a pivotal determinant of Kupffer cell activation in liver carcinogenesis, deepening mechanistic insights into how chronic inflammation underpins the development and progression of liver cancer.

High-throughput molecular and histopathologic profiling of tumor tissue in a novel transplantable model of murine neuroblastoma: new tools for pediatric drug discovery.

Using two MYCN transgenic mouse strains, we established 10 transplantable neuroblastoma cell lines via serial orthotopic passage in the adrenal gland. Tissue arrays demonstrate that by histochemistry, vascularity, immunohistochemical staining for neuroblastoma markers, catecholamine analysis, and concurrent cDNA microarray analysis, there is a close correspondence between the transplantable lines and the spontaneous tumors. Several genes closely associated with the pathobiology and immune evasion of neuroblastoma, novel targets that warrant evaluation, and decreased expression of tumor suppressor genes are demonstrated. These studies describe a unique and generalizable approach to expand the utility of transgenic models of spontaneous tumor, providing new tools for preclinical investigation.

Oncogene-driven intrinsic inflammation induces leukocyte production of tumor necrosis factor that critically contributes to mammary carcinogenesis.

Oncogene activation promotes an intrinsic inflammatory pathway that is crucial for cancer development. Here, we have investigated the actual effect of the inflammatory cytokine tumor necrosis factor (TNF) on the natural history of spontaneous mammary cancer in the HER2/neuT (NeuT) transgenic mouse model. Bone marrow transplantation from TNF knockout mice into NeuT recipients significantly impaired tumor growth, indicating that the source of TNF fostering tumor development was of bone marrow origin. We show that the absence of leukocyte-derived TNF disarranged the tumor vasculature, which lacked pericyte coverage and structural integrity, leading to diffuse vascular hemorrhage and stromal necrosis. In addition, tumor-associated Tie2-expressing monocytes were reduced and cytokine expression skewed from Th2 to Th1 type. Treatment of NeuT mice with anti-TNF antibody partially phenocopied the antitumor effect of TNF-deficient bone marrow cell transplantation, providing a strong preclinical background and rationale for the introduction of TNF antagonists in the treatment of human breast cancer, including basal-like samples for which consolidated targeted therapies do not exist.

MyD88-mediated signaling prevents development of adenocarcinomas of the colon: role of interleukin 18.

Signaling through the adaptor protein myeloid differentiation factor 88 (MyD88) promotes carcinogenesis in several cancer models. In contrast, MyD88 signaling has a protective role in the development of azoxymethane (AOM)/dextran sodium sulfate (DSS) colitis-associated cancer (CAC). The inability of Myd88(-/-) mice to heal ulcers generated upon injury creates an altered inflammatory environment that induces early alterations in expression of genes encoding proinflammatory factors, as well as pathways regulating cell proliferation, apoptosis, and DNA repair, resulting in a dramatic increase in adenoma formation and progression to infiltrating adenocarcinomas with frequent clonal mutations in the beta-catenin gene. Others have reported that toll-like receptor (Tlr) 4-deficient mice have a similar susceptibility to colitis to Myd88-deficient mice but, unlike the latter, are resistant to CAC. We have observed that mice deficient for Tlr2 or Il1r do not show a differential susceptibility to colitis or CAC. However, upon AOM/DSS treatment Il18(-/-) and Il18r1(-/-) mice were more susceptible to colitis and polyp formation than wild-type mice, suggesting that the phenotype of Myd88(-/-) mice is, in part, a result of their inability to signal through the IL-18 receptor. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that impact tissue homeostasis and carcinogenesis.

Immunologic and therapeutic synergy of IL-27 and IL-2: enhancement of T cell sensitization, tumor-specific CTL reactivity and complete regression of disseminated neuroblastoma metastases in the liver and bone marrow.

IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8(+) ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4(+)CD25(+)Foxp3(+) regulatory and IL-17-expressing CD4(+) cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.

Toll-like receptor 9 activation of signal transducer and activator of transcription 3 constrains its agonist-based immunotherapy.

Although toll-like receptor (TLR) agonists, such as CpG, are used as immunotherapeutic agents in clinical trials for cancer and infectious diseases, their effects are limited and the underlying mechanism(s) that restrains CpG efficacy remains obscure. Here, we show that signal transducer and activator of transcription 3 (Stat3) plays a key role in down-modulating immunostimulatory effects of CpG. In the absence of interleukin-6 (IL-6) and IL-10 induction, CpG directly activates Stat3 within minutes through TLR9. Ablating Stat3 in hematopoietic cells results in rapid activation of innate immunity by CpG, with enhanced production of IFN-gamma, tumor necrosis factor-alpha, IL-12, and activation of macrophages, neutrophils, and natural killer cells marked with Stat1 activation. Innate immune responses induced by CpG in mice with a Stat3-ablated hematopoietic system cause potent antitumor effects, leading to eradication of large (>1 cm) B16 melanoma tumors within 72 h. Moreover, ablating Stat3 in myeloid cells increases CpG-induced dendritic cell maturation, T-cell activation, generation of tumor antigen-specific T cells, and long-lasting antitumor immunity. A critical role of Stat3 in mediating immunosuppression by certain cytokines and growth factors in the tumor microenvironment has been recently documented. By demonstrating direct and rapid activation of Stat3 by TLR agonists, we identify a second level of Stat3-mediated immunosuppression. Our results further suggest that targeting Stat3 can drastically improve CpG-based immunotherapeutic approaches.