T cell senescence is thought to contribute to immune function decline, but the pathways that mediate senescence in these cells are not clear. Here, we evaluated T cell populations from healthy volunteers and determined that human CD8+ effector memory T cells that reexpress the naive T cell marker CD45RA have many characteristics of cellular senescence, including decreased proliferation, defective mitochondrial function, and elevated levels of both ROS and p38 MAPK. Despite their apparent senescent state, we determined that these cells secreted high levels of both TNF-α and IFN-γ and showed potent cytotoxic activity. We found that the senescent CD45RA-expressing population engaged anaerobic glycolysis to generate energy for effector functions. Furthermore, inhibition of p38 MAPK signaling in senescent CD8+ T cells increased their proliferation, telomerase activity, mitochondrial biogenesis, and fitness; however, the extra energy required for these processes did not arise from increased glucose uptake or oxidative phosphorylation. Instead, p38 MAPK blockade in these senescent cells induced an increase in autophagy through enhanced interactions between p38 interacting protein (p38IP) and autophagy protein 9 (ATG9) in an mTOR-independent manner. Together, our findings describe fundamental metabolic requirements of senescent primary human CD8+ T cells and demonstrate that p38 MAPK blockade reverses senescence via an mTOR-independent pathway

Highly differentiated CD8 ؉ CD28 ؊ CD27 ؊ T cells have short telomeres, defective telomerase activity, and reduced capacity for proliferation, indicating that they are close to replicative senescence. In addition, these cells express increased levels of the senescence-associated inhibitory receptor KLRG1 and have poor capacity for IL-2 synthesis and defective Akt (ser 473 ) phosphorylation after activation. It is not known whether signaling via KLRG1 contributes to any of the attenuated differentiation-related functional changes in CD8 ؉ T cells. To address this, we blocked KLRG1 signaling during T-cell receptor activation using antibodies against its major ligand, E-cadherin. This resulted in a significant enhancement of Akt (ser 473 ) phosphorylation and T-cell receptor-induced proliferative activity of CD8 ؉ CD28 ؊ CD27 ؊ T cells. Furthermore, the increase of proliferation was directly linked to the Akt-mediated induction of cyclin D and E and reduction in the cyclin inhibitor p27 expression. In contrast, the reduced telomerase activity in highly differentiated CD8 ؉ CD28 ؊ CD27 ؊ T cells was not altered by KLRG1 blockade, indicating the involvement of other mechanisms. This is the first demonstration of a functional role for KLRG1 in primary human CD8 ؉ T cells and highlights that certain functional defects that arise during progressive T-cell differentiation toward replicative senescence are maintained actively by inhibitory receptor signaling. (Blood. 2009;113:6619-6628)

The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8+ T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8+CD28− T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (ζ) chain into primary human CD8+ T cells. Although CD3 ζ-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8+CD28− T cells could up-regulate this enzyme. The further fractionation of CD8+CD28− T cells into CD8+CD28− CD27+ and CD8+CD28−CD27− subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8+CD28−CD27− T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser473 but not the Thr308 phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8+CD28−CD27− T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8+ T cells to be maintained by continuous proliferation in vivo.

CD4؉ CD25 ؉ regulatory T cells have been shown to maintain peripheral tolerance against self and foreign antigens. In this study we analyzed the effect of circulating CD4؉ CD25 ؉ T cells on CD8 ؉ -T-cell responses of patients with chronic and resolved hepatitis B virus (HBV) infection. We demonstrated that circulating CD4 ؉ CD25؉ T cells modulate the function and expansion of HBV-specific CD8 ؉ cells ex vivo in all patients, regardless of whether they have chronic or resolved HBV infection. The possible role of CD4 ؉ CD25 ؉ T cells in the pathogenesis of chronic HBV infection is not supported by these data. However, these results might have implications for optimizing future immunotherapeutic approaches to HBV treatment.Hepatitis B virus (HBV) is a noncytopathic, hepatotropic DNA virus that infects more than 300 million people worldwide, causing liver disease of variable severity (15). The pathogenesis of the liver damage during HBV infection is immune mediated and is dependent on the balance between viral replication and the CD8 ϩ -T-cell response (7). Virus-specific CD8 ϩ cells are necessary for HBV control (25) but are defective in patients with persistent HBV infection compared with those who resolved infection (11). High antigen dose deletion (28) and lack of CD4 help (13) could explain the low number of virus-specific CD8 ϩ cells present in patients with chronic infection. However, it remains possible that other direct mechanisms of regulation of CD8 ϩ expansion operate in patients with chronic HBV infection, who still possess low frequencies of virus-specific CD8 ϩ cells in lymph nodes (18), liver (17), and blood (28). The suggestion that a residual population of cells is actively suppressed in chronic HBV infection is supported by the boosting of their frequencies on reduction of viral load with antiviral therapy (9).Studies with a large number of experimental models have provided convincing evidence that a population of specialized T cells able to actively regulate the immune response represents an integral part of the T-cell repertoire (19,21 CD25ϩ cells to respond to peripheral antigens could be particularly important in the regulation of immunopathological and protective responses to parasites (6) and viruses (1,6,12,24). In mice infected with herpes simplex virus, not only were CD4 ϩ CD25 ϩ cells shown to regulate the clonal expansion of virus-specific CD8 ϩ cells but their suppressive function was also enhanced by the virus infection (24). These data suggest that, during viral infection, CD4ϩ CD25 ϩ cells can be modulated in the periphery after recognition of viral antigens. Furthermore, recent data from hepatitis C virus (HCV)-infected subjects have shown the potential ability of CD4 ϩ CD25 ϩ cells to regulate HCV-specific T cells in patients with chronic hepatitis C (23). These data raise the possibility that a dynamic regulation of virus-specific CD8 ϩ responses can be mediated by CD4ϩ CD25 ϩ cells during viral infection. It is possible that CD4 ϩ CD25 ϩ cells are activated in vivo to suppress ...

a b s t r a c tA novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale threedimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano-and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(e-caprolactone) (PCL) scaffold with modes of fiber distribution at 600 nm and 3.3 lm. Three conventional unimodal scaffolds with mean diameters of 300 nm and 2.6 and 5.2 lm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Young's modulus $40 MPa and strength $1 MPa) in comparison with the controls, despite the large porosity ($90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.

T he Hippo pathway has been recently identified as a crucial axis in the regulation of organ size and shape during organogenesis and cancer. The paralog Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1 or TAZ) are the downstream effectors of the Hippo pathway. These proteins have also been identified as mammalian proto-oncogenes. 1 Moreover, they perform as transcriptional coactivators in the nucleus, mainly in combination with transcription factors belonging to TEAD family. 2 In vitro, YAP/TAZ activity has been associated with mesenchymal stem cell (MSC) fate decision through the interaction with key determinants of osteogenic (Runx2) or adipogenic (PPARγ) differentiation. 3,4 YAP/TAZ axis has also emerged as a central regulator of human embryonic stem cell self-renewal through the control of SMAD complex shuttling to the nucleus, with TAZ knock-down resulting in the loss of cell pluripotency. 5 The same cofactors control intestinal 6 and neural progenitor cell number and differentiation 7 by targeting We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.

No abstract

A number of previous studies investigated the in vitro effects of resveratrol on malignant human breast epithelial cell replication. The aim of the present study was to evaluate the activity of resveratrol on human metastatic breast cancer cells. The study was performed on the MCF-7 tumor cell line. Cell growth, cell cycle perturbation and apoptosis were evaluated by trypan blue dye exclusion assay, flow cytometric analysis and confocal fluorescence microscopy. TRAP assay and Western blot analysis respectively detected levels of telomerase activity and levels of hTERT in intracellular compartments of MCF-7 cells treated with resveratrol. Resveratrol has a direct inhibitory effect on cell proliferation. The results demonstrate that the drug induces apoptosis in MCF-7 cells, in a time-and concentrationrelated manner. Our results also show that the growth-inhibitory effect of resveratrol on malignant cells is mainly due to its ability to induce S-phase arrest and apoptosis in association with reduced levels of telomerase activity. In particular, TRAP assay and Western blot analysis respectively showed that resveratrol treatment down-regulates the telomerase activity of target cells and the nuclear levels of hTERT, the reverse transcriptase subunit of the telomerase complex. In our experimental model of breast cancer, resveratrol shows direct antiproliferative and pro-apoptotic effects. Studies on telomerase function and intracellular hTERT distribution point out that this agent is endowed with additional suppressive functions on critical tumor biological properties. These results speak in favor of a potential role of resveratrol in chemoprevention/chemotherapy of breast cancer.