Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Constitutive p16Ink4a expression, along with senescence-associated β-galactosidase (SAβG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAβG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAβG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAβG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAβG-positive macrophages.

Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated β-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system. In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and MS analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging. Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation.aging | oxidative posttranslational modifications | biomarker | SAMP8 | malondialdehyde

Sustained delivery of IL-12 and GM-CSF to tumors induces the activation of tumor-resident CD8؉ T effector/memory cells (Tem) followed by cytotoxic CD8 ؉ T effector cell expansion. To determine whether the secondary effectors expanded from tumorassociated Tem or were primed de novo, activation kinetics of tumor-draining lymph node (TDLN) CD8 ؉ T cells were analyzed. Treatment promoted a 4-fold increase in the numbers of TDLN CD8؉ T cells displaying a CD69 ؉ CCR5 ؉ CD62L ؊ peripheryhoming effector phenotype by day 4 posttherapy. Pulse labeling of tumor and TDLN T cells with BrdU confirmed that proliferation occurred exclusively within the draining lymph nodes between days 1 and 4 with subsequent migration of primed CD8؉ T effectors to tumors on day 7. Day 4 CD8؉ T effector cells preferentially homed to and lysed experimental, but not control, tumors, establishing tumor specificity. To determine whether the secondary CD8 ؉ T effector cell response was dependent on activation of tumor-resident CD8 ؉ Tem, mice that were selectively depleted of tumor-infiltrating CD8 ؉ T cells were treated and monitored for T effector priming. In the absence of tumor-resident CD8 ؉ Tem, T effector cell expansion was completely abrogated in the TDLN, revealing that restoration of CD8 T he majority of tumor immunotherapy protocols are designed to induce antitumor T cell responses due to the exquisite specificity and potent cytotoxicity of T cells (1). However, such vaccines have generally been ineffective in achieving regression of established tumors in murine tumor models and in cancer patients (2). The observed lack of therapeutic efficacy is not simply due to the qualitative and/or quantitative inferiority of vaccine-induced antitumor T cells but rather appears to be associated with the immune-suppressive characteristics of the tumor microenvironment (3, 4); that is, even when potent and long-lasting tumor-specific T cell activity is generated, vaccine-induced cells are either unable to accumulate in tumors or are inactivated rapidly upon infiltration into tumor (3-6). Functional analyses of either vaccine-induced or natural tumor-infiltrating T cell populations have demonstrated that their quiescent phenotype is primarily associated with signaling defects (7,8). At the same time, others have shown that this phenotype is reversible and that purification and ex vivo culture of tumor-infiltrating T lymphocytes (TIL) 3 can result in the recovery of their cytotoxic function (9, 10). To this end, T cells expanded from tumors have been utilized in adoptive T cell transfer therapy to achieve durable clinical regressions in melanoma patients (11).Whether activation of TIL in situ can overcome tumor-mediated immune suppression and achieve tumor regression has been addressed in several studies. Targeting of inhibitory/regulatory mechanisms in tumors, including selective elimination of T suppressor cells or blocking of coinhibitory molecules can rescue concomitant immunity and lead to tumor regression even in the absence of additional immune st...

Sustained intratumoral delivery of Interleukin-12 (IL-12) and Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) can overcome tumor immune suppression and promote T-cell-dependent eradication of established disease in murine tumor models. However the antitumor effector response is transient and is rapidly followed by a T-suppressor cell rebound. The mechanisms that control the switch from an effector to a regulatory response in this model have not been defined. Since Dendritic cells (DC) can mediate both effector and suppressor T-cell priming, DC activity was monitored in the tumors and the tumor-draining lymph nodes (TDLN) of IL-12/GM-CSF-treated mice. The studies demonstrated that therapy promoted the recruitment of immunogenic DC (iDC) to tumors with subsequent migration to the TDLN within 24 to 48 hours of treatment. Longer-term monitoring revealed that immunogenic DC converted to an indoleamine 2,3 dioxygenase (IDO)-positive tolerogenic phenotype (tDC) in the TDLN between days 2 and 7. Specifically, day 7 DC lost the ability to prime CD8+ T-cells but preferentially induced CD4+ Foxp3+ T-cells. The functional switch was reversible as inhibition of IDO with 1-methyl tryprophan (1-MT) restored immunogenic function to tDC. All post-therapy immunological activity was strictly associated with conventional myeloid DC and no functional changes were observed in the plasmacytoid DC subset throughout treatment. Importantly, the initial recruitment and activation of immunogenic DC as well as the subsequent switch to tolerogenic activity were both driven by IFNγ revealing the dichotomous role of this cytokine in regulating IL-12-mediated antitumor T-cell immunity.