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...