Cellular senescence, a stress-induced irreversible growth arrest often characterized by p16Ink4a expression and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time and have been speculated to play a role in aging. To explore the physiological relevance and consequences of naturally occurring senescent cells, we used a previously established transgene, INK-ATTAC, to induce apoptosis in p16Ink4a-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. Here we show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. Clearance of p16Ink4a-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels, and adipocytes, respectively. Thus, p16Ink4a-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in multiple organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

Evidence suggests that regulatory T cells expressing the transcription factor Foxp3 develop extrathymically and intrathymically. Mechanisms of extrathymic induction require further scrutiny, especially as proliferation and/or phenotypic changes of preexisting suppressor cells must be distinguished from true de novo generation. Here we report the conversion of truly naive CD4(+) T cells into suppressor cells expressing Foxp3 by targeting of peptide-agonist ligands to dendritic cells and by analysis of Foxp3 expression at the level of single cells. We show that conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. The addition of transforming growth factor-beta or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. In addition, regulatory T cell populations induced in subimmunogenic conditions could subsequently be expanded by delivery of antigen in immunogenic conditions. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity.

Cancer patients can harbor significant numbers of CD8 and CD4 T cells with specificities to tumor antigens (Ags). Yet, in most cases, such T cells fail to eradicate the tumor in vivo. Here, we investigated the interference of Ag-specific CD4 ؉ CD25 ؉ regulatory T cells (Treg) with the tumor-specific CD8 T cell immune response in vivo, by monitoring the homing, expansion, and effector function of both subsets in draining and nondraining lymph nodes. The results show that CD8 cells expand to the same extent and produce similar levels of IFN-␥ in the presence or absence of Ag-specific Treg. Nevertheless, these Treg abrogate CD8 T cell-mediated tumor rejection by specifically suppressing the cytotoxicity of expanded CD8 cells. The molecular mechanism of suppression involves TGF-␤ because expression of a dominant-negative TGF-␤ receptor by tumor-specific CD8 cells renders them resistant to suppression and is associated with tumor rejection and unimpaired cytotoxicity. (5-7). Treg also can control the magnitude of recall CD8 T cell responses in different settings that include viral (8, 9) and bacterial (10) infections as well as allograft transplantation in vivo (11,12). A role in Treg function has been attributed to IL-2, which stimulates Treg and in turn may inhibit division of memory CD8 T cells (13,14). However, these studies did not trace Ag-specific Treg in draining lymph nodes (LN), and it is not clear to what extent the different outcomes reflect accumulation of Treg by specific homing and local expansion. In fact, most studies were conducted with polyclonal Treg of unknown specificity where such questions cannot be addressed, and, therefore, the mode of inhibition cannot be correlated with specific Treg accumulation. Analysis of tumor-bearing patients suggests that suppression of CD8 T cell cytotoxicity by Treg may be causally related to tumor progression, because tumor-specific CD8 cells and tumor-specific CD4 Treg frequently accumulate in tumors from melanoma patients, and tumor-specific CD8 cells fail to exert cytotoxic T lymphocyte effector function (15)(16)(17).This study investigates how Treg suppress primary CD8 T cell immune responses directed against tumor cells expressing influenza hemagglutinin (HA) as a surrogate tumor-specific Ag. Naïve CD8 and regulatory CD4 T cells with transgenic receptors specific for distinct peptides of HA were used to allow us to follow the fate of these cells in tumor draining LN. The results show that Treg interfere with CD8 T cell-mediated tumor rejection relatively early during the immune response, and the mechanism by which Treg suppress naïve CD8 cells differs from the ones that have been reported for memory CD8 cells (10)(11)(12)14). Treg influenced neither the kinetics of proliferation nor the commitment of recently activated CD8 cells to produce inflammatory cytokines. Nevertheless, CD8 cells failed to undergo normal functional maturation in the presence of Treg as evidenced by the fact that their cytotoxic potential to destroy specific targets in vivo was abolis...

Mechanisms of dominant tolerance have evolved within the mammalian immune system to prevent inappropriate immune responses. CD4(+)CD25(+) regulatory T (T(reg)) cells have emerged as central constituents of this suppressive activity. By using multiphoton intravital microscopy in lymph nodes (LNs) of anesthetized mice, we have analyzed how cytotoxic T lymphocytes (CTLs) interact with antigen-presenting target cells in the presence or absence of activated T(reg) cells. Nonregulated CTLs killed their targets at a 6.6-fold faster rate than regulated CTLs. In spite of this compromised effector activity, regulated CTLs exhibited no defect in proliferation, induction of cytotoxic effector molecules and secretory granules, in situ motility, or ability to form antigen-dependent conjugates with target cells. Only granule exocytosis by CTLs was markedly impaired in the presence of T(reg) cells. This selective form of regulation did not require prolonged contact between CTLs and T(reg) cells but depended on CTL responsiveness to transforming growth factor-beta. CTLs quickly regained full killing capacity in LNs upon selective removal of T(reg) cells. Thus, T(reg) cells reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill.

Dominant mechanisms of tolerance control the autoimmune potential of self-reactive T cells in healthy individuals and animals (reviewed in refs. 1-3). Insights into the regulation of immune responses by regulatory T cells have been mostly obtained with polyclonal populations of regulatory T cells for which the role of specific antigen has been largely obscure. The impact of self-antigen on the shaping of the regulatory T cell pool came more into focus when it was observed that the coexpression of a transgenic class II MHC restricted T cell receptor (TCR) and its agonist ligand resulted in the generation of antigen-specific regulatory T cells (4). Subsequent experiments in that system confirmed the notion that thymic epithelium can have a decisive role in the formation of such cells by demonstrating that the expression of an agonist ligand on radioresistant tissue (5) and on transplanted thymic epithelium (6) was a very effective means of generating regulatory T cells, whereas the mode of generation of polyclonal regulatory T cells in normal mice still needs to be elucidated.A subset of CD4 ϩ T cells expressing the interleukin-2 (IL-2) receptor ␣-chain (CD25) recently became a major focus of interest. These CD4 ϩ CD25 ϩ T cells were first shown by Sakaguchi and colleagues to control autoreactive T cells in vivo (7). Several characteristics of these cells have emerged from in vitro studies (reviewed in ref. 8) resulting in the notion that regulatory T cells are anergic in terms of proliferation and suppress other cells by direct cell contact, which requires neither IL-10 nor transforming growth factor-␤ and which results in the inability of suppressed CD4 ϩ T cells to produce IL-2 (9-11). However, it is unclear at present how far these observations in vitro are in fact a reflection of the properties of CD4 ϩ CD25 ϩ T cells in vivo. In some experimental systems of immune regulation by CD4 ϩ CD25 ϩ T cells in vivo, it was found that soluble factors such as IL-4, IL-10, and transforming growth factor-␤ do contribute to the prevention of autoimmunity, with the role of these factors var ying between different models (12-14). Polyclonal CD25 ϩ CD4 ϩ T cells proliferate and expand when they are transferred into rag Ϫ/Ϫ or IL-2 receptor ␤-deficient mice (15-17), indicating that their anergic state can be reversed under certain nonphysiological conditions. Notably, it is under exactly these lymphopenic conditions that the regulatory function of CD25 ϩ CD4 ϩ T cells has been studied in the majority of the currently available models, at least suggesting that proliferation and suppressive function by regulatory T cells may not be mutually exclusive. A major experimental drawback of lymphopenic models of immune regulation, however, is that they provide no information on antigen-induced proliferation as observed in nonlymphopenic mice.The present study was initiated to establish an in vivo system of antigen-specific immune regulation that is as physiological as possible to characterize the behavior of CD25 ϩ CD4 ϩ regulatory T ...

Replication-defective retroviruses expressing the t-neu oncogene, or a hybrid protein with the neu tyrosine kinase linked to the external region of the human epidermal growth factor receptor (egfr-neu), were used to establish lines of murine oligodendroglial precursor cells. Differentiation of the t-neu lines into myelin-associated glycoprotein (MAG)-positive oligodendrocytes was induced by dibutyryl cAMP, and the egfr-neu line showed limited differentiation in vitro upon withdrawal of epidermal growth factor. Cerebellar granule cell neurons expressed mitogens for the cell lines. Upon transplantation into demyelinated lesions, t-neu line cells engaged with the demyelinated axons whereas the egfr-neu line cells differentiated further and ensheathed the axons. These cell lines thus interact with neurons in vitro and in vivo and can be used as tools to define the molecules involved in different stages of neuron-glia interaction.

It is generally agreed that most colon cancers develop from adenomatous polyps, and it is this fact on which screening strategies are based. Although there is overwhelming evidence to link intrinsic genetic lesions with the formation of these preneoplastic lesions, recent data suggest that the tumor stromal environment also plays an essential role in this disease. In particular, it has been suggested that CD34 ؉ immature myeloid precursor cells are required for tumor development and invasion. Here we have used mice conditional for the stabilization of ␤-catenin or defective for the adenomatous polyposis coli (APC) gene to reinvestigated the identity and importance of tumor-infiltrating hematopoietic cells in polyposis. We show that, from the onset, polyps are infiltrated with proinflammatory mast cells (MC) and their precursors. Depletion of MC either pharmacologically or through the generation of chimeric mice with genetic lesions in MC development leads to a profound remission of existing polyps. Our data suggest that MC are an essential hematopoietic component for preneoplastic polyp development and are a novel target for therapeutic intervention.cancer ͉ inflammation ͉ polyposis ͉ TNF␣

SummaryThere is a growing acceptance that tumor-infiltrating myeloid cells play an active role in tumor growth and mast cells are one of the earliest cell types to infiltrate developing tumors. Mast cells accumulate at the boundary between healthy tissues and malignancies and are often found in close association with blood vessels within the tumor microenvironment. They express many proangiogenic compounds, and may play an early role in angiogenesis within developing tumors. Mast cells also remodel extracellular matrix during wound healing, and this function is subverted in tumor growth, promoting tumor spread and metastasis. In addition, mast cells modulate immune responses by dampening immune rejection or directing immune cell recruitment, depending on local stimuli. In this review, we focus on key roles for mast cells in angiogenesis, tissue remodeling and immune modulation and highlight recent findings on the integral role that mast cells play in tumor growth. New findings suggest that mast cells may serve as a novel therapeutic target for cancer treatment and that inhibiting mast cell function may lead to tumor regression.