The effector functions of CD8+ T cells are influenced by tissue inflammatory microenvironments. IL-33, a member of the IL-1 family, acts as a danger signal after its release during cell necrosis. The IL-33/ST2 axis has been implicated in various Th2 responses. Its role in CD8+ T cell-mediated immune response is, however, not known. Here we find that type 1 cytotoxic T (Tc1) cells cultured in vitro unexpectedly express high levels of the IL-33 receptor ST2. Interestingly, the expression of ST2 in Tc1 cells is dependent on T-bet, a master Th1/Tc1 transcription factor. In addition, IL-33 enhances TCR-triggered IFN-γ production. IL-33 together with IL-12 can stimulate IFN-γ production in Tc1 cells. Moreover, IL-33 synergizes with IL-12 to promote CD8+ T cell effector function. The synergistic effect of IL-33 and IL-12 is partly mediated by Gadd45b. Together, these in vitro data establish a novel role of IL-33 in promoting effector type 1 adaptive immune responses.

Background

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model.

Methods

CD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.

Results

The engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.

Conclusion

CNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8+ T cells in both chronic infection and tumor. However, the nature of TIM-3+CD4+ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4+ and CD8+ TILs from human lung cancer tissues but negligibly expressed on T cells from patients' peripheral blood. Frequencies of IFN-γ+ cells were reduced in TIM-3+CD8+ TILs compared to TIM-3−CD8+ TILs. However, the level of TIM-3 expression on CD8+ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3+CD4+ TILs expressed FOXP3 and about 60% of FOXP3+ TILs were TIM-3+. Importantly, TIM-3 expression on CD4+ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.

Background

The goal of this study is to evaluate the effectiveness of S-1/Oxaliplatin vs. Doxifluridine/Oxaliplatin regimen and to identify miRNAs as potential prognostic biomarkers in gastric cancer patients. The expression of candidate miRNAs was quantified from fifty-five late stage gastric cancer FFPE specimens.

Experimental Design

Gastric cancer patients with KPS>70 were recruited for the trial. The control group was treated with 400 mg/twice/day Doxifluridine plus i.v. with Oxaliplatin at 130 mg/m2/first day/4 week cycle. The testing group was treated with S-1 at 40 mg/twice/day/4 week cycle plus i.v. with Oxaliplatin at 130 mg/m2/first day/4 week cycle. Total RNAs were extracted from normal and gastric tumor specimens. The levels of miRNAs were quantified using real time qRT-PCR expression analysis.

Results

The overall objective response rate (CR+PR) of patients treated with S-1/Oxaliplatin was 33.3% (CR+PR) vs. 17.6% (CR+PR) with Doxifluridine/Oxaliplatin for advanced stage gastric cancer patients. The average overall survival for patients treated with S-1/Oxaliplatin was 7.80 month vs. 7.30 month with patients treated with Doxifluridine/Oxaliplatin. The expression of miR-181b (P = 0.022) and miR-21 (P = 0.0029) was significantly overexpressed in gastric tumors compared to normal gastric tissues. Kaplan-Meier survival analysis revealed that low levels of miR-21 expression (Log rank test, hazard ratio: 0.17, CI = 0.06–0.45; P = 0.0004) and miR-181b (Log rank test, hazard ratio: 0.37, CI = 0.16–0.87; P = 0.018) are closely associated with better patient's overall survival for both S-1 and Doxifluridine based regimens.

Conclusion

Patients treated with S-1/Oxaliplatin had a better response than those treated with Doxifluridine/Oxaliplatin. miR-21 and miR-181b hold great potential as prognostic biomarkers in late stage gastric cancer.

Summary

An effective Th1 type cell-mediated immune response against cancer cells is critical in limiting cancer progression. Gadd45b, a signaling molecule highly upregulated during Th1 type responses, is studied for its role in limiting tumor growth. Mouse B16 melanoma cells implanted into Gadd45b−/− mice grew faster than those in wild type or Gadd45b +/− littermate controls. The defect of Gadd45b−/− mice in tumor immunosurveillance was attributed to the reduced expression of IFN-γ, granzyme B, and CCR5 in Gadd45b−/− CD8+ T cells at the tumor site. Activation of p38 MAP kinase, but not ERK or JNK, by either TCR-stimuli or IL-12 and IL-18 is diminished in Gadd45b−/− CD8+ T cells, resulting in reduced production of IFN-γ. In addition, mRNA of T-bet and Eomes were reduced in Gadd45b−/− CD8+ T cells, supporting a critical role of Gadd45b in shaping the Th1 fate. More importantly, the tumor vaccination which is effective in wild type mice failed in Gadd45b/Gadd45g doubly deficient mice. Collectively, these data demonstrate that members of the Gadd45 gene family are important for anti-tumor immune responses.

Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4–/– bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4–/– BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.

Background

Phenobarbital (PB) is the most well-known among numerous non-genotoxic carcinogens that cause the development of hepatocellular carcinoma (HCC). PB activates nuclear xenobiotic receptor Constitutive Active/Androstane Receptor (CAR; NR1I3) and this activation is shown to determine PB promotion of HCC in mice. The molecular mechanism of CAR-mediated tumor promotion, however, remains elusive at the present time. Here we have identified Growth Arrest and DNA Damage-inducible 45β (GADD45B) as a novel CAR target, through which CAR represses cell death.

Methodology/Principal Findings

PB activation of nuclear xenobiotic receptor CAR is found to induce the Gadd45b gene in mouse liver throughout the development of HCC as well as in liver tumors. Given the known function of GADD45B as a factor that represses Mitogen-activated protein Kinase Kinase 7 - c-Jun N-terminal Kinase (MKK7-JNK) pathway-mediated apoptosis, we have now demonstrated that CAR interacts with GADD45B to repress Tumor Necrosis Factor α ( TNFα)-induced JNK1 phosphorylation as well as cell death. Primary hepatocytes, prepared from Car+/+, Car−/−, Gadd45b+/+ and Gadd45b−/− mice, were treated with TNFα and Actinomycin D to induce phosphorylation of JNK1 and cell death. Co-treatment with the CAR activating ligand TCPOBOP (1,4 bis[2-(3,5-dichloropyridyloxy)]benzene) has resulted in repression of both phosphorylation and cell death in the primary hepatocytes from Car+/+ but not Car−/−mice. Repression by TCPOBOP was not observed in those prepared from Gadd45b−/− mice. In vitro protein-protein interaction and phosphorylation assays have revealed that CAR interacts with MKK7 and represses the MKK7-mediated phosphorylation of JNK1.

Conclusions/Significance

CAR can form a protein complex with GADD45B, through which CAR represses MKK7-mediated phosphorylation of JNK1. In addition to activating the Gadd45b gene, CAR may repress death of mouse primary hepatocytes by forming a GADD45B complex and repressing MKK7-mediated phosphorylation of JNK1. The present finding that CAR can repress cell death via its interaction with GADD45B provides an insight for further investigations into the CAR-regulated molecular mechanism by which PB promotes development of HCC.

Recent studies have established an important role of Th17 in induction of autoimmune diseases. We have found that although IL-17 receptor A (IL-17RA) −/− mice were resistant to experimental autoimmune encephalomyelitis (EAE), a small number of them developed milder clinical signs of this autoimmune disease. In addition, blockade of TGFβ in IL-17RA−/− mice resulted in much more severe clinical signs of EAE and significantly increased parenchymal lymphocyte infiltration in the central nervous system (CNS). Furthermore, the number of autoreactive Th1 cells was greatly increased in the inflamed spinal cord of IL-17RA−/− mice. These data support a role of IL-17RA-independent mechanisms in causing autoimmunity and its regulation by TGFβ.

The mammalian brain exhibits diverse types of neural plasticity, including activity-dependent neurogenesis in the adult hippocampus. How transient activation of mature neurons leads to long-lasting modulation of adult neurogenesis is unknown. Here we identify Gadd45b as a neural activity–induced immediate early gene in mature hippocampal neurons. Mice with Gadd45b deletion exhibit specific deficits in neural activity–induced proliferation of neural progenitors and dendritic growth of newborn neurons in the adult hippocampus. Mechanistically, Gadd45b is required for activity-induced DNA demethylation of specific promoters and expression of corresponding genes critical for adult neurogenesis, including brain-derived neurotrophic factor and fibroblast growth factor. Thus, Gadd45b links neuronal circuit activity to epigenetic DNA modification and expression of secreted factors in mature neurons for extrinsic modulation of neurogenesis in the adult brain.

The number of effector T cells is controlled by proliferation and programmed cell death. Loss of these controls on self-destructive effector T cells may precipitate autoimmunity. Here, we show that two members of the growth arrest and DNA damage-inducible (Gadd45) family, β and γ, are critical in the development of pathogenic effector T cells. CD4+ T cells lacking Gadd45β can rapidly expand and invade the central nervous system in response to myelin immunization, provoking an exacerbated and prolonged autoimmune encephalomyelitis in mice. Importantly, mice with compound deficiency in Gadd45β and Gadd45γ spontaneously developed signs of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus. Our findings therefore identify the Gadd45β/Gadd45γ-mediated control of effector autoimmune lymphocytes as an attractive novel target for autoimmune disease therapy.

Fibrosis and apoptosis are juxtaposed in pulmonary disorders such as asthma and the interstitial diseases, and transforming growth factor (TGF)-β1 has been implicated in the pathogenesis of these responses. However, the in vivo effector functions of TGF-β1 in the lung and its roles in the pathogenesis of these responses are not completely understood. In addition, the relationships between apoptosis and other TGF-β1–induced responses have not been defined. To address these issues, we targeted bioactive TGF-β1 to the murine lung using a novel externally regulatable, triple transgenic system. TGF-β1 produced a transient wave of epithelial apoptosis that was followed by mononuclear-rich inflammation, tissue fibrosis, myofibroblast and myocyte hyperplasia, and septal rupture with honeycombing. Studies of these mice highlighted the reversibility of this fibrotic response. They also demonstrated that a null mutation of early growth response gene (Egr)-1 or caspase inhibition blocked TGF-β1–induced apoptosis. Interestingly, both interventions markedly ameliorated TGF-β1–induced fibrosis and alveolar remodeling. These studies illustrate the complex effects of TGF-β1 in vivo and define the critical role of Egr-1 in the TGF-β1 phenotype. They also demonstrate that Egr-1–mediated apoptosis is a prerequisite for TGF-β1–induced fibrosis and remodeling.

The Rho subfamily of small GTP-binding proteins mediates many fundamental cellular functions. The commonly studied members (Rho, Rac, and CDC42) regulate actin reorganization, affecting diverse cellular responses, including adhesion, cytokinesis, and motility. Another major function of the Rho GTPases is their role in regulating transcriptional factors and nuclear signaling. RhoH is encoded by a hematopoiesis-specific Rho-related gene recently identified in a fusion transcript with bcl6 in lymphoma cell lines. Significantly, translocations and a high frequency of RhoH mutation have been detected in primary lymphoma cells. We show here that RhoH functions differently from other Rho GTPases. RhoH exerts no significant effect on actin reorganization. However, RhoH is a potent inhibitor of the activation of NFκB and p38 by other Rho GTPases. This property, together with the differential expression of RhoH in the Th1 subset of T cells, suggests a role for RhoH in the functional differentiation of T cells. RhoH has different amino acids in two highly conserved residues critical for GTPase activity. Consequently, RhoH is GTPase deficient, remaining in a GTP-bound activated state without cycling. Reduction of RhoH levels in T cells augments the response to Rac activation. Furthermore, RhoH is dramatically down regulated after phorbol myristate acetate treatment and in Th1 cells after activation by anti-CD3. Hence, a mechanism for regulation of RhoH function is likely to exist at the transcriptional level. The inhibitory function of RhoH supports a model in which Rho GTPases with opposing functions may compete to modulate the final outcome of a particular GTPase-activated pathway.

One mechanism regulating the ability of different subsets of T helper (Th) cells to respond to cytokines is the differential expression of cytokine receptors. For example, Th2 cells express both chains of the interferon γ receptor (IFN-γR), whereas Th1 cells do not express the second chain of the IFN-γR (IFN-γR2) and are therefore unresponsive to IFN-γ. To determine whether the regulation of IFN-γR2 expression, and therefore IFN-γ responsiveness, is important for the differentiation of naive CD4+ T cells into Th1 cells or for Th1 effector function, we generated mice in which transgenic (TG) expression of IFN-γR2 is controlled by the CD2 promoter and enhancer. CD4+ T cells from IFN-γR2 TG mice exhibit impaired Th1 polarization potential in vitro. TG mice also display several defects in Th1-dependent immunity in vivo, including attenuated delayed-type hypersensitivity responses and decreased antigen-specific IFN-γ production. In addition, TG mice mount impaired Th1 responses against Leishmania major, as manifested by increased parasitemia and more severe lesions than their wild-type littermates. Together, these data suggest that the sustained expression of IFN-γR2 inhibits Th1 differentiation and function. Therefore, the acquisition of an IFN-γ–unresponsive phenotype in Th1 cells plays a crucial role in the development and function of these cells.