Reciprocal costimulatory molecules control the activation of mucosal type 3 innate lymphoid cells during engagement with B cells.

Innate lymphoid cells (ILCs) are the counterpart of T helper cells in the innate immune system and share multiple phenotypes with T helper cells. Inducible T-cell costimulator (ICOS) is recognized on T cells and participates in T-cell activation and T and B-cell engagement in lymphoid tissues. However, the role of ICOS in ILC3s and ILC3-involved interactions with the immune microenvironment remains unclear. Here, we found that ICOS expression on human ILC3s was correlated with the activated state of ILC3s. ICOS costimulation enhanced the survival, proliferation, and capacity of ILC3s to produce cytokines (IL-22, IL-17A, IFN-γ, TNF, and GM-CSF). Via synergistic effects of ICOS and CD40 signaling, B cells promoted ILC3 functions, and ILC3-induced T-cell-independent B-cell IgA and IgM secretion primarily required CD40 signaling. Hence, ICOS is essential for the nonredundant role of ILC3s and their interaction with adjacent B cells.

Correlation Between Immune Lymphoid Cells and Plasmacytoid Dendritic Cells in Human Colon Cancer.

Background: Innate lymphoid cells (ILCs), so far studied mostly in mouse models, are important tissue-resident innate immune cells that play important roles in the colorectal cancer microenvironment and maintain mucosal tissue homeostasis. Plasmacytoid dendritic cells (pDCs) present complexity in various tumor types and are correlated with poor prognosis. pDCs can promote HIV-1-induced group 3 ILC (ILC3) depletion through the CD95 pathway. However, the role of ILC3s in human colon cancer and their correlation with other immune cells, especially pDCs, remain unclear. Methods: We characterized ILCs and pDCs in the tumor microenvironment of 58 colon cancer patients by flow cytometry and selected three patients for RNA sequencing. Results: ILC3s were negatively correlated, and pDCs were positively correlated, with cancer pathological stage. There was a negative correlation between the numbers of ILC3s and pDCs in tumor tissues. RNA sequencing confirmed the correlations between ILC3s and pDCs and highlighted the potential function of many ILC- and pDC-associated differentially expressed genes in the regulation of tumor immunity. pDCs can induce apoptosis of ILC3s through the CD95 pathway in the tumor-like microenvironment. Conclusions: One of the interactions between ILC3s and pDCs is via the CD95 pathway, which may help explain the role of ILC3s in colon cancer.

GDF15 promotes glioma stem cell-like phenotype via regulation of ERK1/2-c-Fos-LIF signaling.

Growth differentiation factor 15 (GDF15), a member of the transforming growth factor ß family, is associated with tumor progression, metastasis, and cell apoptosis. However, controversy persists regarding the role of GDF15 in different tumor types, and its function in glioma stem cells (GSCs) remains unknown. Here, we report that GDF15 promotes the GSC-like phenotype in GSC-like cells (GSCLCs) through the activation of leukemia inhibitor factor (LIF)-STAT3 signaling. Mechanistically, GDF15 was found to upregulate expression of the transcription factor c-Fos, which binds to the LIF promoter, leading to enhanced transcription of LIF in GSCLCs. Furthermore, GDF15 may activate the ERK1/2 signaling pathway in GSCLCs, and the upregulation of LIF expression and the GSC-like phenotype was dependent on ERK1/2 signaling. In addition, the small immunomodulator imiquimod induced GDF15 expression, which in turn activated the LIF-STAT3 pathway and subsequently promoted the GSC-like phenotype in GSCLCs. Thus, our results demonstrate that GDF15 can act as a proliferative and pro-stemness factor for GSCs, and therefore, it may represent a potential therapeutic target in glioma treatment.

Discrepant antitumor efficacies of three CpG oligodeoxynucleotide classes in monotherapy and co-therapy with PD-1 blockade.

Unmethylated CpG oligodeoxynucleotides (ODNs) activate plasmacytoid dendritic cells (pDCs) and B cells to induce humoral and cellular immunity, and are under development for the treatment of multiple cancers. However, the specific differences in antitumor effects among the three CpG ODN classes when administered as a monotherapy or in co-therapy with the anti-PD-1 antibody are unclear. We compared the immunostimulatory effects in vitro and antitumor effects in a CT26 subcutaneous mouse tumor model among the three CpG ODN classes. We found that CpG-A slightly suppressed tumor growth but possessed no synergistic antitumor effects with the anti-PD-1 antibody. CpG-B at low doses significantly inhibited tumor growth and possessed synergistic antitumor effects with the anti-PD-1 antibody. A high dose of CpG-C was required to achieve antitumor effects comparable to those of CpG-B, which was consistent with the immunostimulatory effects in B-cell proliferation and TLR9-NF-κB activation. Importantly, CpG-C in combination with anti-PD-1 antibody inhibited tumor growth more quickly and effectively than CpG-B because CpG-B significantly upregulated PD-L1 expression on multiple host immune cells to promote tumor immune escape. Moreover, co-therapy increased the infiltration of effector memory T cells. In summary, CpG-B and CpG-C with different optimal concentrations possessed strong antitumor effects, while CpG-C was more rapid and effective for co-therapy with the anti-PD-1 antibody.

A Dual TLR7/TLR9 Inhibitor HJ901 Inhibits ABC-DLBCL Expressing the MyD88 L265P Mutation.

Diffuse large B cell lymphoma (DLBCL) is associated with aggressive clinical cases and poor prognosis despite recent advances in disease treatment. In activated B-cell-like (ABC)-DLBCL, the most severe damaged signaling pathways converge to aberrantly activate the Toll-like receptor (TLR) 7/9/MyD88 pathways, leading to the avoidance of cell death and resistance to chemotherapy. A gain of function mutation in MyD88 (MyD88 L265P) enhanced the NF-κB and JAK-STAT signaling pathways and was associated with dysregulation of TLR signaling in the pathogenesis of ABC-DLBCL. Therefore, inhibition of the TLR signaling network may improve clinical outcomes. In this study, we designed a de novo synthesized oligodeoxynucleotide-based antagonist of TLR7 and TLR9, referred to as HJ901, which competitively binds to TLR7/9. We profiled HJ901 inhibition in various DLBCL cell lines and verified tumor suppression in a xenograft mouse model. We found that HJ901 treatment significantly reduced TLR7- and TLR9-mediated cell proliferation and cytokine production in a time- and dose-dependent manner in various DLBCL cell lines expressing the MyD88 L265P mutation. Moreover, HJ901 prevented tumor growth and downregulated the NF-κB and JAK2-STAT3 signaling pathways in a DLBCL xenograft mouse model with the MyD88 L265P mutation. These results reveal that the anti-tumor effects of the synthesized oligodeoxynucleotide-based antagonist, HJ901, which competitively binds to TLR7/9, may be associated with the downregulation of the NF-κB and JAK2-STAT3 signaling pathways and provide rationale for treating ABC-DLBCL patients with the MyD88 L265P mutation.

A Novel C Type CpG Oligodeoxynucleotide Exhibits Immunostimulatory Activity In Vitro and Enhances Antitumor Effect In Vivo.

BACKGROUND: C type CpG oligodeoxynucleotides (CpG-C ODNs), possessing the features of both A type and B type CpG ODNs, exert a variety of immunostimulatory activities and have been demonstrated as an effective antitumor immunotherapy. Based on the structural characteristics, we designed 20 potential ODNs with the aim of synthesizing an optimal, novel CpG-C ODN specific to human and murine Toll-like receptor 9 (TLR9). We also sought to investigate the in vitro immunostimulatory and in vivo antitumor effects of the novel CpG-C ODN. METHODS: Twenty potential CpG-C ODNs were screened for their ability to secrete interferon (IFN)-α, and interleukin (IL)-6 and tumor necrosis factor (TNF)-α production for the three most promising sequences were assayed in human peripheral blood mononuclear cells (PBMCs) by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array assay. The functions of human and mouse B cells, and cytokine production in mice induced by the most promising sequence, HP06T07, were determined by flow cytometry and ELISA. Growth and morphology of tumor tissues in in vivo murine models inoculated with CT26 cells were analyzed by a growth inhibition assay and immunohistochemistry, respectively. RESULTS: Among the 20 designed ODNs, HP06T07 significantly induced IFN-α, IL-6, and TNF-α secretion, and promoted B-cell activation and proliferation in a dose-dependent manner in human PBMCs and mouse splenocytes in vitro. Intratumoral injection of HP06T07 notably suppressed tumor growth and prolonged survival in the CT26 subcutaneous mouse model in a dose-dependent manner. HP06T07 administered nine times at 2-day intervals (I2) eradicated tumor growth at both primary and distant sites of CT26 tumors. HP06T07 restrained tumor growth by increasing the infiltration of T cells, NK cells, and plasmacytoid dendritic cells (pDCs). CONCLUSIONS: HP06T07, a novel CpG-C ODN, shows potent immunostimulatory activity in vitro and suppresses tumor growth in the CT26 subcutaneous mouse model.

Human Cancer Cells Sense Cytosolic Nucleic Acids Through the RIG-I-MAVS Pathway and cGAS-STING Pathway.

Pattern recognition receptors (PRRs) are germline-encoded host sensors of the innate immune system. Some human cancer cells have been reported to express PRRs. However, nucleic acid sensors in human cancers have not been studied in detail. Therefore, we systematically analyzed the expression, molecular cascade, and functions of TLR3, RIG-I, MDA5, LGP2, cGAS, and STING in human cancer cells. TLR3, TRIF, RIG-I, MDA5, LGP2, and MAVS were expressed in 22 cell lines. The majority of cell lines responded to only RIG-I ligands 5'-ppp-dsRNA, Poly(I:C)-HMW, Poly(I:C)-LMW, and/or Poly(dA:dT), as revealed by IRF3 phosphorylation and IFN-ß secretion. IFN-ß secretion was inhibited by RIG-I and MAVS knockdown. cGAS and STING were co-expressed in 10 of 22 cell lines, but IFN-ß secretion was not induced by STING ligands ISD, HSV60, VACV70, Poly(dG:dC), and 3'3'-cGAMP in cGAS and STING intact cell lines. Further experiments revealed that the cGAS-STING pathway was activated, as revealed by TBK1 and IRF3 phosphorylation and IFN-ß and ISG mRNA expression. These results suggest that human epithelial cancer cells respond to cytosolic RNA through the RIG-I-MAVS pathway but only sense cytosolic DNA through the cGAS-STING pathway. These findings are relevant for cancer immunotherapy approaches based on targeting nucleic acid receptors.

pDC Activation by TLR7/8 Ligand CL097 Compared to TLR7 Ligand IMQ or TLR9 Ligand CpG.

Plasmacytoid dendritic cells (pDCs) express high levels of the toll-like receptors (TLRs) TLR7 and TLR9. In response to TLR7 and TLR9 ligands, pDCs are primary producers of type I interferons. Our previous study demonstrated that pDCs activated by the TLR7 ligand imiquimod (IMQ) and the TLR9 ligand CpG A can kill breast cancer cells in vitro and inhibit tumor growth in vivo. Moreover, we observed a distinctive morphological, phenotypic change in pDCs after activation by IMQ and CpG A. However, the effect of other TLR7 and TLR9 ligands on pDCs remains less understood. In this study, we treat pDCs with the TLR7 ligand IMQ, TLR7/8 ligands (CL097 and CL075), and three TLR9 ligands (different types of CpGs). The size of pDCs increased significantly after activation by TLR7, or TLR7/8 ligands. TLR7, TLR7/8, and TLR9 ligands similarly modulated cytokine release, as well as protein expression of pDC markers, costimulatory molecules, and cytotoxic molecules. Interestingly, TLR7/8 ligands, especially CL097, induced stronger responses. These results are relevant to the further study of the role and mechanism of pDC-induced antitumor effects and may aid in the development of a new strategy for future tumor immunotherapy.

An effective dendritic cell-based vaccine containing glioma stem-like cell lysate and CpG adjuvant for an orthotopic mouse model of glioma.

Owing to the limited therapeutic efficacy of glioma vaccines, new strategies are required to improve cancer vaccines. Our study aimed to assess the therapeutic efficacy of a glioma vaccine called STDENVANT. This vaccine, comprising glioma stem-like cell (GSC) lysate, dendritic cells (DCs), and Toll-like receptor (TLR) 9 agonist CpG motif-containing oligodeoxynucleotides (CpG ODNs), was assessed using a GL261-C57BL/6 orthotopic mouse model of glioma. STDENVANT markedly improved survival and tumor regression by enhancing anti-tumor immune function. Moreover, STDENVANT upregulated programmed death 1 (PD-1) and its ligand PD-L1 on effector T cells, DCs, and glioma tissues, resulting in the accumulation of regulatory T (Treg) cells in the brain and lymph nodes. Combinatorial administration of anti-PD-L1 antibody and STDENVANT conferred a greater survival advantage and decreased the Treg cell population in the brain. The present results indicate that PD-L1 blockade can promote tumor regression via STDENVANT in a mouse model of glioma, and combinatorial administration of anti-PD-L1 antibody and STDENVANT increases the therapeutic anti-tumor efficacy of treatment.