Penghui Zhou

Dendritic cells in the thymus contribute to T-regulatory cell induction

Central tolerance is established through negative selection of self-reactive thymocytes and the induction of T-regulatory cells (TRs). The role of thymic dendritic cells (TDCs) in these processes has not been clearly determined. In this study, we demonstrate that in vivo, TDCs not only play a role in negative selection but in the induction of TRs. TDCs include two conventional dendritic cell (DC) subtypes, CD8loSirpαhi/+ (CD8loSirpα+) and CD8hiSirpαlo/− (CD8loSirpα−), which have different origins. We found that the CD8hiSirpα+ DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8loSirpα+ DCs demonstrate a superior capacity to induce TRs in vitro. Finally, using a thymic transplantation system, we demonstrate that the DCs in the periphery can migrate into the thymus, where they efficiently induce TR generation and negative selection.

Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation

CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.

Laforin Negatively Regulates Cell Cycle Progression through Glycogen Synthase Kinase 3β-Dependent Mechanisms

ABSTRACT Glycogen synthase kinase 3β (GSK-3β) represses cell cycle progression by directly phosphorylating cyclin D1 and indirectly regulating cyclin D1 transcription by inhibiting Wnt signaling. Recently, we reported that the Epm2a-encoded laforin is a GSK-3β phosphatase and a tumor suppressor. The cellular mechanism for its tumor suppression remains unknown. Using ex vivo thymocytes and primary embryonic fibroblasts from Epm2a−/− mice, we show here a general function of laforin in the cell cycle regulation and repression of cyclin D1 expression. Moreover, targeted mutation of Epm2a increased the phosphorylation of Ser9 on GSK-3β while having no effect on the phosphorylation of Ser21 on GSK-3α. In the GSK-3β+/+ but not the GSK-3β−/− cells, Epm2a small interfering RNA significantly enhanced cell growth. Consistent with an increased level of cyclin D1, the phosphorylation of retinoblastoma protein (Rb) and the levels of Rb-E2F-regulated genes cyclin A, cyclin E, MCM3, and PCNA are also elevated. Inhibitors of GSK-3β selectively increased the cell growth of Epm2a+/+ but not of Epm2a−/− cells. Taken together, our data demonstrate that laforin is a selective phosphatase for GSK-3β and regulates cell cycle progression by GSK-3β-dependent mechanisms. These data provide a cellular basis for the tumor suppression activity of laforin.

Targeting lymphotoxin-mediated negative selection to prevent prostate cancer in mice with genetic predisposition

The identification of individuals genetically susceptible to cancer calls for preventive measures to minimize the cancer risk in these high-risk populations. Immune prevention is made necessary by the anticipated health threat, but lack of enough high-affinity T cells against tumor-associated antigens and the unpredictability of tumor antigens make antigen-based immune prevention untenable for cancer. To address this issue, we explored a non-antigen-based cancer immune prevention strategy using the transgenic adenocarcinoma of mouse prostate model that spontaneously develops prostate cancer with 100% penetrance. We show that targeted mutation of the lymphotoxin α (LTα) gene efficiently rescued tumor-reactive T cells, drastically reduced cancer incidence, and almost completely ablated metastasis. Remarkably, short-term treatments with the fusion protein consisting of constant region of IgG and extracellular domain of lymphotoxin β receptor (LTβRIg) interrupted clonal deletion, reduced the size of the primary cancer, and completely prevented metastasis later in life. Our data demonstrated the value of non-antigen-based immune prevention for those with a genetic predisposition to cancer.

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells.

CD4+CD25+ regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4+CD25+ T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.

B7 Blockade Alters the Balance between Regulatory T cells and Tumor-reactive T Cells for Immunotherapy of Cancer

Purpose: In prostate cancer-bearing host, regulatory T (Treg) cells restrain activity of tumor antigen-specific T cells. Because B7:CD28 interactions are needed for both function of CD4+CD25+ Treg cells and CD8+ effective T cells, targeting this pathway may help to overcome the immunotherapy barriers. Experimental Design: The anti-B7-1/B7-2 monoclonal antibodies were administered to a transgenic mouse model of prostate cancer (TRAMP) ectopically expressing SV40 large T antigen in different tumor development stages for prevention and therapy of prostate cancer. The treatment was also tested in treating transplanted MC38 colon adenocarcinoma in mice. Results: Here, we showed that short-term administration of anti-B7-1/B7-2 monoclonal antibodies in TRAMP mice leads to significant inhibited primary tumor growth and the size of metastatic lesions. The treatment is effective to inhibit MC38 colon cancer growth. Correspondingly, this treatment results in a transient reduction of Treg in both thymus and the periphery. In vivo cytotoxicity assay revealed T antigen-specific CTL effectors in anti-B7-treated but not control IgG-treated TRAMP mice. Conclusions: Transient blockade of B7-1/B7-2 alters the balance between Treg and cancer-reactive T cells to enhance cancer immunotherapy.

Abstract 4759: Immune prevention of prostate cancer in TRAMP mice targeting lymphotoxin signaling pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC One of the most important advances in cancer research is the identification of individuals with increased susceptibility to cancer. Genetic susceptibility can be conferred by inactive alleles of tumor suppressor genes or by hypermorphic alleles of oncogenes. In extreme cases, inactivating mutations of tumor suppressor genes such as p53, adenomatous polyposis coli (APC) gene and BRCA1/2 can result in a nearly 80% lifelong cancer risk. Recent genome-wide association studies have allowed the identification of numerous cancer susceptibility loci. It is anticipated that increasing numbers of individuals will be diagnosed with high cancer risk, providing an enormous need for the identification of cancer preventative therapies. Although prophylactic surgery and chemoprevention exist as viable options for the prevention of cancer, prophylactic surgery is difficult to implement to those yet to develop cancer given the vital importance of many organs, and chemoprevention has a high burden of compliance and drug safety. Immune prevention is made necessary by the anticipated health threat and possible by predictability of antigens carried by pathogens. The classic notion of immune prevention is based on immunization with antigens expressed by the pathogens. However, cancer antigens are poorly defined, unpredictable and more heterogeneous, which makes it considerably more difficult to design antigen-based vaccines. Furthermore, since cancers are derived from normal tissues, most of the high affinity T cells reactive to such peripheral tissue antigens in the cancer cells have been deleted. Lack of high affinity tumor-reactive T cells would in theory make immune prevention difficult to attain. Recent studies have demonstrated that clonal deletion of T-cells reactive to peripheral antigens depends on their expression in thymic medullar epithelial cells. Since tumors are comprised of malignantly transformed cells from normal tissues, and therefore likely express tissue-specific antigens, it is of interest to determine whether these T cells can be rescued for the purpose of immune prevention. Lymphotoxin α (LTα) gene plays a major role in the development and function of medullar epithelial cells, especially in the context of clonal deletion of peripheral antigen-reactive T cells. Blocking lymphotoxin signaling pathway may allow one to rescue tumor-reactive T cells to prevent the development of cancer. Using mice with a targeted mutation of LTα, we reveal a valuable target for rescuing prostate cancer-reactive T cells and a means of cancer immune prevention. More importantly, transient blockade of LTα significantly reduced the size of the prostate cancer tumor and eliminated cancer metastasis in TRAMP mice. To our knowledge, this strategy represents the first non-antigen-based immune prevention for cancer and has potential to be translated into clinical care of patients with high genetic risk for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4759.