Puyue Wang

Pivotal Role of Reduced let-7g Expression in Breast Cancer Invasion and Metastasis

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer.

Vγ4 γδ T Cell-Derived IL-17A Negatively Regulates NKT Cell Function in Con A-Induced Fulminant Hepatitis

Con A-induced fulminant hepatitis is a well-known animal model for acute liver failure. However, the role of γδ T cells in this model is undefined. In this report, using TCR δ−/− mice, we demonstrated a protective role of γδ T cells in Con A-induced hepatitis model. TCR δ−/− mice showed significantly decreased levels of IL-17A and IL-17F in the Con A-treated liver tissue, and reconstitution of TCR δ−/− mice with wild-type (Wt), but not IL-17A−/−, γδ T cells significantly reduced hepatitis, strongly suggesting a critical role of IL-17A in mediating the protective effect of γδ T cells. Interestingly, only Vγ4, but not Vγ1, γδ T cells exerted such a protective effect. Furthermore, depletion of NKT cells in TCR δ−/− mice completely abolished hepatitis, and NKT cells from Con A-challenged liver tissues of TCR δ−/− mice expressed significantly higher amounts of proinflammatory cytokine IFN-γ than those from Wt mice, indicating that γδ T cells protected hepatitis through targeting NKT cells. Finally, abnormal capacity of IFN-γ production by NKT cells of TCR δ−/− mice could only be downregulated by transferring Wt, but not IL-17−/−, Vγ4 γδ T cells, confirming an essential role of Vγ4-derived IL-17A in regulating the function of NKT cells. In summary, our report thus demonstrated a novel function of Vγ4 γδ T cells in mediating a protective effect against Con A-induced fulminant hepatitis through negatively regulating function of NKT cells in an IL-17A–dependent manner, and transferring Vγ4 γδ T cells may provide a novel therapeutic approach for this devastating liver disease.

Regulatory Role of Vγ1 γδ T Cells in Tumor Immunity through IL-4 Production

It has been demonstrated that the two main subsets of peripheral γδ T cells, Vγ1 and Vγ4, have divergent functions in many diseases models. Recently, we reported that Vγ4 γδ T cells played a protective role in tumor immunity through eomesodermin-controlled mechanisms. However, the precise roles of Vγ1 γδ T cells in tumor immunity, especially whether Vγ1 γδ T cells have any interaction with Vγ4 γδ T cells, remain unknown. We demonstrated in this paper that Vγ1 γδ T cells suppressed Vγ4 γδ T cell-mediated antitumor function both in vitro and in vivo, and this suppression was cell contact independent. Using neutralizing anti–IL-4 Ab or IL-4−/− mice, we determined the suppressive factor derived from Vγ1 γδ T cells was IL-4. Indeed, treatment of Vγ4 γδ T cells with rIL-4 significantly reduced expression levels of NKG2D, perforin, and IFN-γ. Finally, Vγ1 γδ T cells produced more IL-4 and expressed significantly higher level of GATA-3 upon Th2 priming in comparison with Vγ4 γδ T cells. Therefore, to our knowledge, our results established for the first time a negative regulatory role of Vγ1 γδ T cells in Vγ4 γδ T cell-mediated antitumor immunity through cell contact-independent and IL-4–mediated mechanisms. Selective depletion of this suppressive subset of γδ T cells may be beneficial for tumor immune therapy.

Naturally Activated Vγ4 γδ T Cells Play a Protective Role in Tumor Immunity through Expression of Eomesodermin

We previously demonstrated that γδ T cells played an important role in tumor immune surveillance by providing an early source of IFN-γ. The precise role of different subsets of γδ T cells in the antitumor immune response, however, is unknown. Vγ1 and Vγ4 γδ T cells are the principal subsets of peripheral lymphoid γδ T cells and they might play distinct roles in tumor immunity. In support of this, we observed that reconstitution of TCRδ−/− mice with Vγ4, but not Vγ1, γδ T cells restored the antitumor response. We also found that these effects were exerted by the activated (CD44high) portion of Vγ4 γδ T cells. We further determined that IFN-γ and perforin are critical elements in the Vγ4-mediated antitumor immune response. Indeed, CD44high Vγ4 γδ T cells produced significantly more IFN-γ and perforin on activation, and showed greater cytolytic activity than did CD44high Vγ1 γδ T cells, apparently due to the high level of eomesodermin (Eomes) in these activated Vγ4 γδ T cells. Consistently, transfection of dominant-negative Eomes in Vγ4 γδ T cells diminished the level of IFN-γ secretion, indicating a critical role of Eomes in the effector function of these γδ T cells. Our results thus reveal distinct functions of Vγ4 and Vγ1 γδ T cells in antitumor immune response, and identify a protective role of activated Vγ4 γδ T cells, with possible implications for tumor immune therapy.

High susceptibility to liver injury in IL-27 p28 conditional knockout mice involves intrinsic interferon-γ dysregulation of CD4+ T cells.

Interleukin (IL)‐27, a newly discovered IL‐12 family cytokine, is composed of p28 and EBI3. In this study, CD11c‐p28f/f conditional knockout mice were generated to delete p28 specifically in dendritic cells (DCs). We demonstrated that in the absence of DC‐derived p28, these mice were highly susceptible to both low and higher concentrations of concanavalin A (ConA) (5 mg/kg or 10 mg/kg), with extremely early and steady high levels of interferon‐γ (IFN‐γ) in sera. Neutralizing IFN‐γ prevented ConA‐induced liver damage in these mice, indicating a critical role of IFN‐γ in this pathological process. Interestingly, the main source of the increased IFN‐γ in CD11c‐p28f/f mice was CD4+ T cells, but not natural killer T (NKT) cells. Depletion of CD4+, but not NK1.1+, cells completely abolished liver damage, whereas transferring CD4+ T cells from CD11c‐p28f/f mice, but not from wild‐type mice or CD11c‐p28f/f‐IFN‐γ−/− double knockout mice to CD4−/− mice, restored the increased liver damage. Further studies defined higher levels of IFN‐γ and T‐bet messenger RNA in naïve CD4+ T cells from CD11c‐p28f/f mice, and these CD4+ T cells were highly responsive to both low and higher concentrations of anti‐CD3, indicating a programmed functional alternation of CD4+ T cells. Conclusion: We provide a unique model for studying the pathology of CD4+ T cell–mediated liver injury and reveal a novel function of DC‐derived p28 on ConA‐induced fulminant hepatitis through regulation of the intrinsic ability for IFN‐γ production by CD4+ T cells. (HEPATOLOGY 2013)

Therapeutic effects of a novel tylophorine analog, NK‐007, on collagen‐induced arthritis through suppressing tumor necrosis factor α production and Th17 cell differentiation

OBJECTIVE To analyze the effects of a novel compound, NK-007, on the prevention and treatment of collagen-induced arthritis (CIA) and the underlying mechanisms. METHODS We determined the effect of NK-007 on lipopolysaccharide (LPS)-triggered tumor necrosis factor α (TNFα) production by murine splenocytes and a macrophage cell line (RAW 264.7) by enzyme-linked immunosorbent assay, intracellular cytokine staining, and Western blotting. The LPS-boosted CIA model was adopted, and NK-007 or vehicle was administered at different time points after immunization. Mice were monitored for clinical severity of arthritis, and joint tissues were used for histologic examination, cytokine detection, and immunohistochemical staining. Finally, stability of TNFα production and Th17 cell differentiation were studied using quantitative polymerase chain reaction and flow cytometry. RESULTS NK-007 significantly suppressed LPS-induced TNFα production in vitro. Administration of NK-007 completely blocked CIA development and delayed its progression. Furthermore, treatment with NK-007 at the onset of arthritis significantly inhibited the progress of joint inflammation. Administration of NK-007 also suppressed production of TNFα, interleukin-6 (IL-6), and IL-17A in the joint and reduced percentages of IL-17+ cells among CD4+ and γ/δ T cells in draining lymph nodes. We further demonstrated that NK-007 acted on the stability of TNFα messenger RNA and reduced Th17 cell differentiation. In addition, it significantly inhibited levels of IL-6 and IL-17A in human coculture assay. CONCLUSION For its effects on the development and progression of CIA and for its therapeutic effect on CIA, NK-007 has great potential to be a therapeutic agent for human rheumatoid arthritis.

Critical Role of Dendritic Cell–Derived IL-27 in Antitumor Immunity through Regulating the Recruitment and Activation of NK and NKT Cells

Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene–induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell–dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Glycoproteomic analysis of tissues from patients with colon cancer using lectin microarrays and nanoLC-MS/MS

The current study evaluated the glycoproteomic profile of tissues from colon cancer patients. The lectin microarray was first performed to compare the glycoprotein profiles between colon cancer and matched normal tissues. Level of N-acetylglucosamine (GlcNAc) that Solanum tuberosum lectin (STL) bound was found to be elevated in colon cancer, which was verified through lectin histochemistry. The subsequent glycoproteomic analysis based on STL enrichment of glycoproteins followed by label-free quantitative nano liquid chromatography-mass spectrometry/mass spectrometry (nanoLC-MS/MS) analysis identified 72 proteins in high confidence. Among these proteins, 17 were exclusively detected in cancer tissues, and 14 were significantly upregulated in tumor tissues. Annexin A1 and HSP90β were chosen for further investigation by immunoprecipitation coupled with lectin blots, western blots and tissue microarrays. Both Annexin A1 and HSP90β were GlcNAcylated, and their protein expressions were elevated in colon cancer, compared to normal tissues. Moreover, specific changes of GlcNAc abundances in Annexin A1 and HSP90β suggested that tumor-specific glycan patterns could serve as candidate biomarkers of colon cancer for distinguishing cancer patients from healthy individuals.

Current progress in γδ T-cell biology

T lymphocytes bearing γ- and δ-chain T-cell receptor heterodimers are named γδ T cells. Interestingly, γδ and αβ T cells share the same progenitors, and they undergo a fate decision in the thymus. Functional differentiation of γδ T cells occurs both inside and outside the thymus. Antigen recognition of γδ T-cell receptors is very unique, and the responses frequently exhibit innate characteristics. Nevertheless, peripheral γδ T cells exert a number of effector and regulatory functions. γδ T cells rapidly produce cytokines like interferon (IFN)-γ and IL-17 and promote inflammation, partly due to the inherent epigenetic and transcriptional programs, which facilitates a quick and extensive response. Moreover, γδ T cells lyse target cells directly, and this is necessary for pathogen or tumor clearance. γδ T cells can even serve as regulatory cells, and may contribute to immune suppression. Orchestration of γδ T-cell and other immune cell interactions may be critical for host defense and immune regulation. Recently, γδ T cells have been used for immunotherapy for infectious diseases and malignancy. In this review, we summarize the abstracts presented at the recent γδ T cell Conference held from 19 to 21 May 2010, in Kiel, Germany (please see the website for details: http://www.gammadelta-conference.uni-kiel.de/index.html).

A requirement of dendritic cell-derived interleukin-27 for the tumor infiltration of regulatory T cells

Tregs (Foxp3+CD4+) are enriched in tumors to foster a tolerant microenvironment that inhibits antitumor immune response. IL‐27 is reported to regulate the development and function of Tregs in vitro and in vivo; however, the effects of endogenous IL‐27 on Tregs in the tumor microenvironment remain elusive. We demonstrated that in the absence of DC‐derived IL‐27, Tregs were decreased significantly in transplanted B16 melanoma, transplanted EL‐4 lymphoma, and MCA‐induced fibrosarcoma by using IL‐27p28 conditional KO mice. Further studies revealed that IL‐27 promoted the expression of CCL22, which is established to mediate the recruitment of peripheral Tregs into tumors. Tumor‐associated DCs were identified as the major source of CCL22 in tumor sites, and IL‐27 could induce CCL22 expression in an IL‐27R‐dependent manner. Intratumoral reconstitution of rmCCL22 or rmIL‐27, but not rmIL‐27p28, significantly restored the tumor infiltration of Tregs in IL‐27p28 KO mice. Correlated with a decreased number of Tregs, tumor‐infiltrating CD4 T cells were found to produce much more IFN‐γ in IL‐27p28 KO mice, which highlighted the physiological importance of Tregs in suppressing an antitumor immune response. Overall, our results identified a novel mechanism of action of IL‐27 on Tregs in the context of cancers.