Zhihai Qin

CD4+ T Cell–Mediated Tumor Rejection Involves Inhibition of Angiogenesis that Is Dependent on IFNγ Receptor Expression by Nonhematopoietic Cells

Immunity against MHC class II tumors can be mediated by CD4+ T cells in the effector phase through an unknown mechanism. We show that this is IFN gamma dependent but does not require IFN gamma receptor (IFN gamma R) expression on tumor cells, T cells, or other hematopoietic cells and that IFN gamma R expression is not necessary in the priming phase. However, tumor immunity requires IFN gamma R expression on nonhematopoietic cells in the effector phase and involves inhibition of tumor-induced angiogenesis. This shows that an effective anti-tumor response involves communication between CD4+ T cells and nonhematopoietic cells, most likely within the tumor stroma, and that tumor immunity must not entirely rely on direct tumor cell killing.

Involvement of ERK, p38 and NF‐κB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Toll‐like receptors (TLR) are sentinel receptors capable of recognizing pathogen‐associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG‐containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram‐positive and Gram‐negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up‐regulation of co‐stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up‐regulated following LPS stimulation. The up‐regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor‐α induced by LPS plus CpG ODN. While inhibition of extracellular signal‐related kinase and NF‐κB activation suppressed the up‐regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up‐regulation of TLR2 and TLR4 mRNA expression but enhanced the up‐regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up‐regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.

Activation of STAT6 by STING Is Critical for Antiviral Innate Immunity

STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser(407) by TBK1 and Tyr(641), independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6(-/-) mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum.

TNF signaling drives myeloid-derived suppressor cell accumulation

TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor-deficient (Tnfr-/-) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell-mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr-/- mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.

The Gfi-1B Proto-Oncoprotein Represses p21WAF1 and Inhibits Myeloid Cell Differentiation

ABSTRACT Gfi-1 is a cellular proto-oncogene that was identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Here we show that Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. IL-6-induced G1 arrest and differentiation of the myelomonocytic cell line M1 were linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21 WAF1 . Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes revealed that Gfi-1B represses p21WAF1 directly by binding to a high-affinity site at −1518 to −1530 in thep21WAF1 promoter. Forced expression ofGfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocked the IL-6-mediated induction of p21 WAF1 and inhibited G1 arrest and differentiation. We conclude that Gfi-1B is a direct repressor of thep21WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G1 arrest and differentiation of M1 cells perhaps because it prevents p21 WAF1 induction by IL-6.

The role of IFN-γ in tumor transplantation immunity and inhibition of chemical carcinogenesis

IFN-gamma contributes to the rejection of transplantable tumors and the inhibition of methylcholanthrene (MCA)-induced carcinogenesis by different mechanisms. In most tumor transplantation models, tumor rejection requires IFN-gamma receptor expression by host cells, but not by tumor cells. IFN-gamma produced by either CD4+ or CD8+ T cells acts on non-hematopoietic tumor stroma cells and, either directly or indirectly, induces angiostasis. This prevents rapid tumor burden and allows residual tumor cells to be eliminated. In some models, IFN-gamma also contributes to the destruction of existing tumor blood vessels. During MCA-induced tumorigenesis IFN-gamma is involved in the inhibition of MCA diffusion by encapsulation and reduction of DNA damage. This mechanism may primarily protect tissue from damage and simultaneously inhibit tumor development.

Up-regulation of TLR9 gene expression by LPS in mouse macrophages via activation of NF-κB, ERK and p38 MAPK signal pathways

Toll-like receptors (TLR) are critical in the activation of macrophages by bacterial products. It has been shown that TLR2 and TLR4 mediate lipopolysaccharide (LPS) and lipoproteins signal transduction, respectively. Regulation of TLR2 and TLR4 expression by LPS was considered to be one of the mechanisms to control the overall responses of immune cells to bacteria. However, little is known about whether the other members of TLR family are regulated by LPS. Recently, TLR9 was demonstrated to be essential for CpG DNA signaling. Given the effective immune modulation by CpG DNA, regulation of TLR9 expression might play important role in controlling the overall responses of immune cells to bacteria. In this study, regulation of TLR9 gene expression in mouse macrophage cell line RAW264.7 by LPS was investigated. Semiquantitative RT-PCR was performed to determine gene expression of TLR9. Following LPS stimulation, TLR9 gene expression was upregulated within 1 h and reached peak level at about 3 h. LPS stimulation activated NF-kappaB, ERK and p38 MAPK signal pathways. Pretreatment of macrophages with inhibitors of NF-kappaB, ERK and p38 MAPK signal pathways inhibited LPS-induced upregulation of TLR9 mRNA expression. Our results demonstrated that LPS stimulation could upregulate gene expression of TLR9 via NF-kappaB, ERK, and p38 MAPK signal pathways in macrophages, indicating that macrophages with increased TLR9 expression induced by LPS might respond to invading bacteria more effectively.

Inhibition of Methylcholanthrene-induced Carcinogenesis by an Interferon γ Receptor–dependent Foreign Body Reaction

The foreign body reaction is one of the oldest host defense mechanisms against tissue damage which involves inflammation, scarring, and encapsulation. The chemical carcinogen methylcholanthrene (MCA) induces fibrosarcoma and tissue damage in parallel at the injection site. Tumor development induced by MCA but not due to p53-deficiency is increased in interferon-γ receptor (IFN-γR)–deficient mice. In the absence of IFN-γR, MCA diffusion and DNA damage of surrounding cells is increased. Locally produced IFN-γ induces the formation of a fibrotic capsule. Encapsulated MCA can persist virtually life-long in mice without inducing tumors. Together, the foreign body reaction against MCA prevents malignant transformation, probably by reducing DNA damage. This mechanism is more efficient in the presence of IFN-γR. Our results indicates that inflammation and scarring, both suspected to contribute to malignancy, prevent cancer in certain situations.

IFNγ Promotes Papilloma Development by Up-regulating Th17-Associated Inflammation

IFNgamma plays a crucial role in immunity against a variety of transplanted tumors and methylcholanthrene-mediated tumorigenesis in mice. However, it is not clear whether and how endogenous IFNgamma influences 7,12-dimethylbenz(a)anthracene (DMBA)-induced and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papilloma development. We found here that IFNgamma expression was markedly up-regulated shortly after DMBA/TPA application to the skin. Surprisingly, neutralizing IFNgamma activity in vivo did not increase but rather decreased tumor development. Furthermore, IFNgamma receptor-deficient mice were also more resistant to papilloma development than their counterparts were. IFNgamma acted mainly in the promotion stage of papilloma development by enhancing TPA-induced leukocyte infiltration and epidermal hyperproliferation. The up-regulation of tumor necrosis factor alpha, interleukin (IL)-6, and transforming growth factor beta was largely dependent on host IFNgamma responsiveness. Remarkably, up-regulation of both IL-17 expression in the skin and T helper 17 (Th17) cell number in draining lymph nodes after DMBA/TPA treatment was dependent on IFNgamma signaling. Depletion of IL-17 not only decreased the DMBA/TPA-induced inflammation and keratinocyte proliferation but also delayed papilloma development. These results show that IFNgamma, under certain conditions, may promote tumor development by enhancing a Th17-associated inflammatory reaction.

Transmembrane TNF-α Promotes Suppressive Activities of Myeloid-Derived Suppressor Cells via TNFR2

It has been reported that TNFR2 is involved in regulatory T cell induction and myeloid-derived suppressor cell (MDSC) accumulation, two kinds of immunosuppressive cells contributing to tumor immune evasion. Because transmembrane TNF-α (tmTNF-α) is the primary ligand for TNFR2, we hypothesized that tmTNF-α is mainly responsible for the activation of MDSCs. Indeed, we found that tmTNF-α, rather than secretory TNF-α (sTNF-α), activated MDSCs with enhanced suppressive activities, including upregulating arginase-1 and inducible NO synthase transcription, promoting secretion of NO, reactive oxygen species, IL-10, and TGF-β, and enhancing inhibition of lymphocyte proliferation. This effect of tmTNF-α was mediated by TNFR2, as TNFR2 deficiency significantly impaired tmTNF-α–induced release of IL-10 and NO and inhibition of T cell proliferation by MDSC supernatant. Furthermore, tmTNF-α caused p38 phosphorylation and NF-κB activation, whereas inhibition of NF-κB or p38 with an inhibitor pyrrolidine dithiocarbamate or SB203580 abrogated tmTNF-α–mediated increased suppression of lymphocyte proliferation by MDSCs. Consistently, our in vivo study showed that ectopic expression of uncleavable tmTNF-α mutant by 4T1 cells significantly promoted tumor progression and angiogenesis, accompanied with more accumulation of MDSCs and regulatory T cells in the tumor site, increased production of NO, IL-10, and TGF-β, as well as poor lymphocyte infiltration. In contrast, enforced expression of sTNF-α mutant by 4T1 cells that only released sTNF-α without expression of surface tmTNF-α markedly reduced MDSC accumulation and induced more lymphocyte infiltration instead, showing obvious tumor regression. Our data suggest that tmTNF-α acts as a potent activator of MDSCs via TNFR2 and reveals another novel immunosuppressive effect of this membrane molecule that promotes tumor immune escape.