LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

Development of a novel modified vaccine (TheraVacM) for curative treatment of mouse solid tumors.

Monotherapy with immune checkpoint blockade (ICB) antibodies (anti-CTLA4 and anti-PD1/PDL-1) is only effective for 20% to 30% of patients with certain cancers. Patients with cancers harboring few effector T cells (Teffs) are insensitive to ICB therapy. The lack of tumor-specific Teffs is predominantly caused by the paralysis of tumor-infiltrating dendritic cells (TiDCs) resulting from immunosuppression in the tumor microenvironment. We have identified a potent combination of high mobility group nucleosome binding domain 1 (HMGN1, N1) and fibroblast stimulating lipopeptide-1 (FSL-1) that can synergistically trigger maturation of both mouse and human DCs. Accordingly, we designed a combinational anti-cancer immunotherapy with two arms: an immune-activating arm consisting of N1 and FSL-1 to stimulate the generation of Teffs by triggering full maturation of TiDCs, and an ICB arm using anti-PDL-1 or anti-CTLA4 to prevent Teffs from being silenced in the tumor tissue. This combinational immunotherapeutic vaccination regimen dubbed modified TheraVac (TheraVacM) has proved particularly effective as it cured 100% of mice bearing established ectopic CT26 colon and RENCA kidney tumors. The resultant tumor-free mice were resistant to subsequent re-challenge with the same tumors, indicating the generation of long-term tumor specific protective immunity. Since the immune-activating arm also induces full maturation of human DCs, and anti-PDL-1 or anti-CTLA4 have been FDA-approved, this combinational immunotherapy has the potential to be an effective clinical therapy for patients with solid tumors.

Regression and Eradication of Triple-Negative Breast Carcinoma in 4T1 Mouse Model by Combination Immunotherapies.

Triple-negative breast carcinoma (TNBC) is one of the most aggressive types of solid-organ cancers. While immune checkpoint blockade (ICB) therapy has significantly improved outcomes in certain types of solid-organ cancers, patients with immunologically cold TNBC are afforded only a modest gain in survival by the addition of ICB to systemic chemotherapy. Thus, it is urgently needed to develop novel effective therapeutic approaches for TNBC. Utilizing the 4T1 murine model of TNBC, we developed a novel combination immunotherapeutic regimen consisting of intratumoral delivery of high-mobility group nucleosome binding protein 1 (HMGN1), TLR2/6 ligand fibroblast-stimulating lipopeptide (FSL-1), TLR7/8 agonist (R848/resiquimod), and CTLA-4 blockade. We also investigated the effect of adding SX682, a small-molecule inhibitor of CXCR1/2 known to reduce MDSC trafficking to tumor microenvironment, to our therapeutic approach. 4T1-bearing mice responded with significant tumor regression and tumor elimination to our therapeutic combination regimen. Mice with complete tumor regressions did not recur and became long-term survivors. Treatment with HMGN1, FSL-1, R848, and anti-CTLA4 antibody increased the number of infiltrating CD4+ and CD8+ effector/memory T cells in both tumors and draining lymph nodes and triggered the generation of 4T1-specific cytotoxic T lymphocytes (CTLs) in the draining lymph nodes. Thus, we developed a potentially curative immunotherapeutic regimen consisting of HMGN1, FSL-1, R848, plus a checkpoint inhibitor for TNBC, which does not rely on the administration of chemotherapy, radiation, or exogenous tumor-associated antigen(s).

A Therapeutic Vaccine in Combination with Cyclic GMP-AMP Cures More Differentiated Melanomas in Mice.

We have identified a combinational immunotherapy termed TheraVac vaccine (TheraVac) that can cure multiple large established mouse tumors, but it failed to cure melanoma in mice. TheraVac consists of an immunostimulating arm containing an agonist (HMGN1 [N1]) for TLR4 and an agonist (R848) for TLR7/8 that synergize to activate tumor-infiltrating dendritic cells (DCs) and promote Th1 immune responses. The second arm uses an immune checkpoint blockade, anti-PDL-1, to diminish tumor-associated immunosuppression. In this study, we investigated supplementation of TheraVac by a stimulator of IFN genes (STING) agonist, cyclic GMP-AMP (cGAMP), because together they synergize in activating DCs and produced more immunostimulating IL-12p70 and TNF-α cytokines. The synergistic activation and maturation of DCs is dependent on the activation of tank binding kinase-1 (TBK1). Treatment of three different melanin-producing mouse melanomas (B16F1, M3, and M4) with intratumoral delivery of cGAMP and TheraVac eradicated 60-80% of these melanomas. Immunoprofiling of M3 tumor treated with TheraVac plus cGAMP showed an increase in CD8+ CTLs and macrophages in the tumor. There was also a marked increase of CD4, CD8 effector and memory T cells and generation of functional tumor-specific CTLs in tumor-draining lymph nodes. The resultant tumor-free mice were selectively resistant to subsequent challenge with the same tumors, indicating long-term tumor-specific protective immunity. Overall, our findings have important implications for clinical trials with a combination of these immunotherapeutics to cure melanin-producing human melanomas, without the need for exogenous tumor Ags and no clear toxic effects in mice.

TNFR2 antagonistic antibody induces the death of tumor infiltrating CD4+Foxp3+ regulatory T cells.

BACKGROUND: TNFR2 expression is a characteristic of highly potent immunosuppressive tumor infiltrating CD4+Foxp3+ regulatory T cells (Tregs). There is compelling evidence that TNF through TNFR2 preferentially stimulates the activation and expansion of Tregs. We and others, therefore, proposed that targeting TNFR2 may provide a novel strategy in cancer immunotherapy. Several studies have shown the effect of TNFR2 antagonistic antibodies in different tumor models. However, the exact action of the TNFR2 antibody on Tregs remained understood. METHOD: TY101, an anti-murine TNFR2 antibody, was used to examine the effect of TNFR2 blockade on Treg proliferation and viability in vitro. The role of TNFR2 on Treg viability was further validated by TNFR2 knockout mice and in the TY101 antagonistic antibody-treated mouse tumor model. RESULTS: In this study, we found that an anti-mouse TNFR2 antibody TY101 could inhibit TNF-induced proliferative expansion of Tregs, indicative of an antagonistic property. To examine the effect of TY101 antagonistic antibody on Treg viability, we treated unfractionated lymph node (L.N.) cells with Dexamethasone (Dex) which was known to induce T cell death. The result showed that TY101 antagonistic antibody treatment further promoted Treg death in the presence of Dex. This led us to find that TNFR2 expression was crucial for the survival of Tregs. In the mouse EG7 lymphoma model, treatment with TY101 antagonistic antibody potently inhibited tumor growth, resulting in complete regression of the tumor in 60% of mice. The treatment with TY101 antagonistic antibody elicited potent antitumor immune responses in this model, accompanied by enhanced death of Tregs. CONCLUSION: This study, therefore, provides clear experimental evidence that TNFR2 antagonistic antibody, TY101, can promote the death of Tregs, and this effect may be attributable to the antitumor effect of TNFR2 antagonistic antibody.

Interleukin-8: An evolving chemokine.

Early in the 1980s several laboratories mistakenly reported that partially purified interleukin-1 (IL-1) was chemotactic for neutrophils. However, further investigations by us, revealed that our purified IL-1 did not have neutrophil chemotactic activity and this activity in the LPS-stimulated human monocyte conditioned media could clearly be separated from IL-1 activity on HPLC gel filtration. This motivated Teizo Yoshimura and Kouji Matsushima to purify the monocyte-derived neutrophil chemotactic factor (MDNCF), present in LPS conditioned media and molecularly clone the cDNA for MDNCF. They found that MDNCF protein (later renamed IL-8, and finally termed CXCL8) is first translated as a precursor form consisting of 99 amino acid residues and the signal peptide is then removed, leading to the secretion and processing of biologically active IL-8 of 72 amino acid form (residues 28-99). There are four cysteine residues forming two disulfide linkage and 14 basic amino acid residues which result in a very basic property for the binding of IL-8 to heparan sulfate-proteoglycan. The IL-8 gene consists of 4 exons and 3 introns. IL-8 is produced by various types of cells in inflammation. The 5'-flanking region of IL-8 gene contains several nuclear factor binding sites, and NF-κB in combination with AP-1 or C/EBP synergistically activates IL-8 gene in response to IL-1 and TNFα. Two receptors exist for IL-8, CXCR1 and CXCR2 in humans, which belong to γ subfamily of GTP binding protein (G-protein) coupled rhodopsin-like 7 transmembrane domain receptors. Rodents express CXCR2 and do not produce IL-8, but produce numerous homologues instead. Once IL-8 binds to the receptor, ß and γ subunits of G-protein are released from Gα (Gαi2 in neutrophils) and activate PI3Kγ, PLCß2/ß3, PLA2 and PLD. Gαi2 inhibits adenyl cyclase to decrease cAMP levels. Small GTPases Ras/Rac/Rho/cdc42/Rap1, PKC and AKT (PKB) exist down-stream of ß and γ subunits and regulate cell adhesion, actin polymerization, membrane protrusion, and eventually cell migration. PLCß activation generates IP3 and induces Ca++ mobilization, DAG generation to activate protein kinase C to lead granule exocytosis and respiratory burst. MDNCF was renamed interleukin 8 (IL-8) at the International Symposium on Novel Neutrophil Chemotactic Activating Polypeptides, London, UK in 1989. The discovery of IL-8 prompted us to also purify and molecularly clone the cDNA of MCAF/MCP-1 responsible for monocyte chemotaxis, and other groups to identify a large family of chemotactic cytokines capable of attracting other types of leukocytes. In 1992, most of the investigators contributing to the discovery of this new family of chemotactic cytokines gathered in Baden, Austria and agreed to name this family "chemokines" and subsequently established the CXCL/CCL and CXCR/CCR nomenclature. The discovery of chemokines resulted in solving the long-time enigma concerning the mechanism of cell type specific leukocyte infiltration into inflamed tissues and provided a molecular basis for immune and hematopoietic cell migration and interactions under physiological as well as pathological conditions. To our surprise based on its recently identified multifunctional activities, IL-8 has evolved from a neutrophil chemoattractant to a promising therapeutic target for a wide range of inflammatory and neoplastic diseases. IL-8 was initially characterized as a chemoattractant of neutrophils engaged in acute inflammation and then discovered to also be chemotactic for endothelial cells with a major role in angiogenesis. These two activities of IL-8 foster its stimulatory effect on tumor growth. This is abetted by recent additional discoveries showing that IL-8 has stimulatory effects on stem cells and can therefore directly promote the growth of receptor expressing cancer stem cells. IL-8 by interacting with bone marrow stem/progenitor cells has also the capacity to mobilize and release hematopoietic cells into the peripheral circulation. This includes the mobilization of neutrophilic myeloid-derived suppressor cells (N-MDSC) to infiltrate into tumors and thus further promotes the immune escape of tumors. Finally, the capacity of IL-8 to induce trans-differentiation of epithelial cancer cells into mesenchymal phenotype (EMT) increases the malignancy of tumors by promoting their metastatic spread and resistance to chemotherapeutics and cytotoxic immune cells. These observations have stimulated considerable current efforts to develop receptor antagonists for IL-8 and humanized anti-IL-8 antibody for the therapy of cancer, particularly in combination with immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies.

Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA.

Synthetic messenger RNA (mRNA) is an emerging therapeutic platform with important applications in oncology and infectious disease. Effective mRNA medicines must be translated by the ribosome but not trigger a strong nucleic acid-mediated immune response. To expand the medicinal chemistry toolbox for these agents, here we report the properties of the naturally occurring nucleobase N4-acetylcytidine (ac4C) in synthetic mRNAs. We find that ac4C is compatible with, but does not enhance, protein production in the context of synthetic mRNA reporters. However, replacement of cytidine with ac4C diminishes inflammatory gene expression in immune cells caused by synthetic mRNAs. Chemoproteomic capture indicates that ac4C alters the protein interactome of synthetic mRNAs, reducing binding to cytidine-binding proteins and an immune sensor. Overall, our studies illustrate the unique ability of ac4C to modulate RNA-protein interactions and provide a foundation for using N4-cytidine acylation to fine-tune the properties of nucleic acid therapeutics.

Alpha synuclein, the culprit in Parkinson disease, is required for normal immune function.

Alpha-synuclein (αS) is causally involved in the development of Parkinson disease (PD); however, its role in normal vertebrate physiology has remained unknown. Recent studies demonstrate that αS is induced by noroviral infection in the enteric nervous system of children and protects mice against lethal neurotropic viral infection. Additionally, αS is a potent chemotactic activator of phagocytes. In this report, using both wild-type and αS knockout mice, we show that αS is a critical mediator of inflammatory and immune responses. αS is required for the development of a normal inflammatory response to bacterial peptidoglycan introduced into the peritoneal cavity as well as antigen-specific and T cell responses following intraperitoneal immunization. Furthermore, we show that neural cells are the sources of αS required for immune competence. Our report supports the hypothesis that αS accumulates within the nervous system of PD individuals because of an inflammatory/immune response.

A TNFR2 antibody by countering immunosuppression cooperates with HMGN1 and R848 immune stimulants to inhibit murine colon cancer.

Immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs) promote tumor immune evasion and thus targeting of Tregs has become an strategy in cancer immunotherapy. Tumor necrosis factor receptor 2 (TNFR2) is highly expressed and important for the immunosuppressive function of Tregs in humans and mice. Thus, the benefit of targeting TNFR2 in cancer immunotherapy merits more investigation. A previous report identified a new murine monoclonal anti-TNFR2 antibody (designated TY101), which showed therapeutic efficacy in murine cancer models, but its mechanism of action was less understood. In this study, the capacity of a combination of immunostimulants to enhance the effect of this inhibitor of Tregs was investigated. We examined the efficacy of TY101 as an anti-tumor immune reagent combined with HMGN1 (N1, a dendritic cell activating TLR4 agonist) and R848 (a synthetic TLR7/8 agonist). This immunotherapeutic combination exerted synergistic antitumor effects as compared with any single treatment. The antitumor response was mainly mediated by the depletion of Tregs and stimulation of cytotoxic CD8 T cell activation. The result also suggested that the effect of TY101 was similar to that of anti-PD-L1 when used in combination with these immunostimulants. Therefore, we propose that treatment strategies of antagonizing TNFR2 on Tregs would behave as potent checkpoint inhibitors and can potentially be utilized to develop a novel antitumor immunotherapy.

Combining an Alarmin HMGN1 Peptide with PD-L1 Blockade Results in Robust Antitumor Effects with a Concomitant Increase of Stem-Like/Progenitor Exhausted CD8+ T Cells.

The expansion of intratumoral stem-like/progenitor exhausted CD8+ T (Tstem/Tpex) cells provides a potential approach to improve the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, here we demonstrate a strategy to facilitate Tstem/Tpex cell expansion by combining an alarmin high-mobility group nucleosome binding domain 1 (HMGN1) peptide with programmed death-ligand 1 (PD-L1) blockade. The antitumor effects of HMGN1, anti-PD-L1, and their combined treatment were monitored in the B16F10, LLC, Colon26, or EO771 tumor-bearing mice. The comprehensive immunologic analyses, such as high-dimensional flow cytometry, transcriptome analysis, and single-cell RNA-sequencing (scRNA-seq), were used to investigate the cellular and molecular mechanisms of antitumor immune responses after treatments. We identified the immunostimulatory domain (EPKRR SARLS AKPPA KVEAK PKK) on HMGN1 and synthesized this domain as a therapeutic peptide (minP1). Combined treatment with minP1 and PD-L1 blockade induced durable tumor regression in tumor-bearing mice. minP1 increased the number of intratumoral mature DCs enriched in immunoregulatory molecules (mregDC) and enhanced their MHC class I antigen-presenting program. minP1 also synergized with PD-L1 blockade in augmenting intratumoral Tstem/Tpex cell number. Analysis of our scRNA-seq dataset by CellPhonDB suggested potential interactions between mregDCs and Tstem/Tpex cells in tumors. Our results indicate that HMGN1 peptide (minP1) serves as an immunoadjuvant to promote effective anti-PD-L1 immunotherapy with increased Tstem/Tpex cells in tumors.

Inhibition of two-pore channels in antigen-presenting cells promotes the expansion of TNFR2-expressing CD4+Foxp3+ regulatory T cells.

CD4+Foxp3+ regulatory T cells (Tregs) are pivotal for the inhibition of autoimmune inflammatory responses. One way to therapeutically harness the immunosuppressive actions of Tregs is to stimulate the proliferative expansion of TNFR2-expressing CD4+Foxp3+ Tregs via transmembrane TNF (tmTNF). Here, we report that two-pore channel (TPC) inhibitors markedly enhance tmTNF expression on antigen-presenting cells. Furthermore, injection of TPC inhibitors including tetrandrine, or TPC-specific siRNAs in mice, increases the number of Tregs in a tmTNF/TNFR2-dependent manner. In a mouse colitis model, inhibition of TPCs by tetrandrine markedly attenuates colon inflammation by expansion of Tregs Mechanistically, we show that TPC inhibitors enhance tmTNF levels by disrupting surface expression of TNF-α-converting enzyme by regulating vesicle trafficking. These results suggest that the therapeutic potential of TPC inhibitors is mediated by expansion of TNFR2-expressing Tregs and elucidate the basis of clinical use in the treatment of autoimmune and other inflammatory diseases.

Inhibition of murine hepatoma tumor growth by cryptotanshinone involves TLR7-dependent activation of macrophages and induction of adaptive antitumor immune defenses.

Cryptotanshinone (CT), a purified compound initially isolated from the dried roots of Salvia militorrhiza. Bunge, exhibits cytotoxic antitumor effects on many tumors. We have shown that CT possesses the dual capacities to concomitantly inhibit the proliferation of lung cancer cells and promote the generation of antitumor immunity. In this study, we investigated whether CT could be used to treat hepatocellular carcinoma (HCC) using a mouse Hepa1-6 model. CT inhibited the proliferation of mouse hepatoma (Hepa1-6) cells in vitro by inducing Hepa1-6 cells apoptosis through the JAK2/STAT3 signaling pathway. In addition, CT activated macrophages and polarized mouse bone marrow-derived macrophages (BMM) toward an M1 phenotype in vitro, which depended on the TLR7/MyD88/NF-κB signaling pathway. Furthermore, CT significantly inhibited the growth of syngeneic Hepa1-6 hepatoma tumors, and, in combination with anti-PD-L1 cured Hepa1-6-bearing mice with the induction of long-term anti-Hepa1-6 specific immunity. Immunoprofiling of treated Hepa1-6-bearing mice revealed that CT-promoted activation of tumor-infiltrating macrophages and dendritic cells, induction of antitumor T cell response, and infiltration of effector/memory CD8 T cells in the tumor tissue. Importantly, the immunotherapeutic effects of CT and anti-PD-L1 depended on the presence of CD8 T cells. Thus, CT and anti-PD-L1 may provide an effective immunotherapeutic regimen for human HCC based on a combination of cytotoxic effects and induction of tumor-specific immunity.

Cryptotanshinone has curative dual anti-proliferative and immunotherapeutic effects on mouse Lewis lung carcinoma.

Lung cancer is currently the leading cause of cancer-related mortality with very limited effective therapy. Screening of a variety of traditional Chinese medicines (TCMs) for their capacity to inhibit the proliferation of human lung cancer A549 cells and to induce the in vitro maturation of human DCs led to the identification of cryptotanshinone (CT), a compound purified from the TCM Salvia miltiorrhiza Bunge. Here, CT was shown to inhibit the proliferation of mouse Lewis lung carcinoma (LLC) cells by upregulating p53, downregulating cyclin B1 and Cdc2, and, consequently, inducing G2/M cell-cycle arrest of LLC cells. In addition, CT promoted maturation of mouse and human DCs with upregulation of costimulatory and MHC molecules and stimulated DCs to produce TNFα, IL-1ß, and IL-12p70, but not IL-10 in vitro. CT-induced maturation of DCs depended on MyD88 and also involved the activation of NF-κB, p38, and JNK. CT was effective in the treatment of LLC tumors and, when used in combination with low doses of anti-PD-L1, cured LLC-bearing mice with the induction of subsequent anti-LLC long-term specific immunity. CT treatment promoted T-cell infiltration and elevated the expression of genes typical of Th1 polarization in LLC tumor tissue. The therapeutic effect of CT and low doses of anti-PD-L1 was reduced by depletion of CD4 and CD8 T cells. This paper provides the first report that CT induces immunological antitumor activities and may provide a new promising antitumor immunotherapeutic.

Combined treatment with HMGN1 and anti-CD4 depleting antibody reverses T cell exhaustion and exerts robust anti-tumor effects in mice.

BACKGROUND: Transient depletion of CD4+ T cells results in tumor suppression and survival benefit in murine models; however, the tumor progression and recurrence still occur over more long-term monitoring of mice. Thus, we explored an additional strategy to enhance endogenous immune responses by an alarmin, high mobility group nucleosome binding protein 1 (HMGN1). METHODS: The anti-tumor effects of HMGN1, anti-CD4 depleting antibody, and their combined treatment were monitored in the Colon26 or the B16F10 subcutaneous murine models. The tumor-infiltrating CD8+ T cell proliferation, differentiation, exhaustion, and its gene expression were determined by flow cytometry, transcriptome analysis, and quantitative real-time PCR. RESULTS: Our results show that a systemic administration of low doses of HMGN1 with an anti-CD4 depleting antibody (HMGN1/αCD4) promoted expansion of CD8+ T cell populations (e.g. CD137+ PD-1+ and CD44hi PD-1+), recruited CCR7+ migratory dendritic cells to the tumor, and reduced co-inhibitory molecules (e.g. PD-1, LAG-3, and TIM-3) to counteract CD8+ T cell exhaustion. CONCLUSION: The HMGN1/αCD4 treatment expanded effector CD8+ T cells and prolonged their anti-tumor activities by rescuing them from exhaustion, thus resulting in tumor regression and even rejection in long-term monitored mice.

High-mobility group nucleosome binding domain 1 (HMGN1) functions as a Th1-polarizing alarmin.

High-mobility group (HMG) nucleosome binding domain 1 (HMGN1), which previously was thought to function only as a nucleosome-binding protein that regulates chromatin structure, histone modifications, and gene expression, was recently discovered to be an alarmin that contributes extracellularly to the generation of innate and adaptive immune responses. HMGN1 promotes DC recruitment through interacting with a Gαi protein-coupled receptor (GiPCR) and activates DCs predominantly through triggering TLR4. HMGN1 preferentially promotes Th1-type immunity, which makes it relevant for the fields of vaccinology, autoimmunity, and oncoimmunology. Here, we discuss the alarmin properties of HMGN1 and update recent advances on its roles in immunity and potential applications for immunotherapy of tumors.

High-Mobility Group Nucleosome-Binding Protein 1 as Endogenous Ligand Induces Innate Immune Tolerance in a TLR4-Sirtuin-1 Dependent Manner in Human Blood Peripheral Mononuclear Cells.

High-mobility group nucleosome-binding protein 1 (HMGN1) functions as a non-histone chromatin-binding protein in the cell nucleus. However, extracellular HMGN1 acts as an endogenous danger-associated inflammatory mediator (also called alarmin). We demonstrated that HMGN1 not only directly stimulated cytokine production but also had the capacity to induce immune tolerance by a TLR4-dependent pathway, similar to lipopolysaccharide (LPS)-induced tolerance. HMGN1-induced tolerance was accompanied by a metabolic shift associated with the inhibition of the induction of Warburg effect (aerobic glycolysis) and histone deacetylation via Sirtuin-1. In addition, HMGN1 pre-challenge of mice also downregulated TNF production similar to LPS-induced tolerance in vivo. In conclusion, HMGN1 is an endogenous TLR4 ligand that can induce both acute stimulation of cytokine production and long-term tolerance, and thus it might play a modulatory role in sterile inflammatory processes such as those induced by infection, trauma, or ischemia.

Blockade of TNFR2 signaling enhances the immunotherapeutic effect of CpG ODN in a mouse model of colon cancer.

Through the tumor necrosis factor (TNF) receptor type II (TNFR2), TNF preferentially activates, expands, and promotes the phenotypic stability of CD4+Foxp3+ regulatory T (Treg) cells. Those Treg cells that have a high abundance of TNFR2 have the maximal immunosuppressive capacity. We investigated whether targeting TNFR2 could effectively suppress the activity of Treg cells and consequently enhance the efficacy of cancer immunotherapy. We found that, relative to a suboptimal dose of the immunostimulatory Toll-like receptor 9 ligand CpG oligodeoxynucleotide (ODN), the combination of the suboptimal dose of CpG ODN with the TNFR2-blocking antibody M861 more markedly inhibited the growth of subcutaneously grafted mouse CT26 colon tumor cells. This resulted in markedly fewer TNFR2+ Treg cells and more interferon-γ-positive (IFN-γ+) CD8+ cytotoxic T lymphocytes infiltrating the tumor and improved long-term tumor-free survival in the mouse cohort. Tumor-free mice were resistant to rechallenge by the same but not unrelated (4T1 breast cancer) cells. Treatment with the combination of TNFR2-blocking antibody and a CD25-targeted antibody also resulted in enhanced inhibition of tumor growth in a syngeneic 4T1 mouse model of breast cancer. Thus, the combination of a TNFR2 inhibitor and an immunotherapeutic stimulant may represent a more effective treatment strategy for various cancers.

The Future of the Cytokine Discipline.

The study of cytokines has evolved from the detection of functional activities present in tissue culture supernatants to the characterization of the three-dimensional molecular structures of the cytokines and their receptors. Investigators studying cytokines need to have specialized expertise in using cytokine assays, assessing their receptor interactions, signal transduction, gene activation, and biological effects, and in the therapeutic utilization of agonists and antagonists. Cytokinology can therefore be considered a discipline. In this article, I have considered studies leading to the identification of novel cytokines, potential producers of cytokine mimics such as viruses and the microbiome, and the complex interactions of the cytokine network with our vital functions. Our ever-increasing success in using cytokines and, in particular, cytokine inhibitors therapeutically suggest that cytokinology will eventually become an independent discipline.

HMGN1 and R848 Synergistically Activate Dendritic Cells Using Multiple Signaling Pathways.

High mobility group nucleosome-binding protein 1 (HMGN1 or N1) is a Th1-polarizing alarmin, but alone is insufficient to induce antitumor immunity. We previously showed that combination of N1 and R848, a synthetic TLR7/8 agonist, synergistically activates dendritic cells (DCs) and induces therapeutic antitumor immunity, however, it remained unclear how N1 and R848 synergistically activate DCs. Here, we show that co-stimulation with N1 and R848 of human monocyte-derived DCs (MoDCs) markedly upregulated DC's surface expression of CD80, CD83, CD86, and HLA-DR, as well as synergistic production of pro-inflammatory cytokines including IL-12p70, IL-1ß, and TNF-α. This combination also synergistically activated NF-κB and multiple MAPKs that are involved in DC maturation. Moreover, N1 and R848 synergistically increased nuclear translocation of interferon (IFN) regulatory transcription factors (e.g., IRF3 and IRF7) and promoted the expression of type 1 IFNs such as IFN-α2, IFN-α4, and IFN-ß1. Similar signaling pathways were also induced in mouse bone marrow-derived DCs (BMDCs). RNA-seq analysis in human MoDCs revealed that N1 plus R848 synergistically upregulated the expression of genes predominantly involved in DC maturation pathway, particularly genes critical for the polarization of Th1 immune responses (e.g., IL12A, IL12B, and IFNB1, etc.). Overall, our findings show that (1) N1 synergizes with R848 in activating human and mouse DCs and (2) the synergistic effect based on various intracellular signaling events culminated in the activation of multiple transcriptional factors. These findings have important implications for future clinical trials since N1 and R848 synergistically promoted optimal Th1 lineage immune responses resulting in tumor rejection in mice.