A chemogenomic screening identifies CK2 as a target for pro-senescence therapy in PTEN-deficient tumours

Enhancement of cellular senescence in tumours triggers a stable cell growth arrest and activation of an antitumour immune response that can be exploited for cancer therapy. Currently, there are only a limited number of targeted therapies that act by increasing senescence in cancers, but the majority of them are not selective and also target healthy cells. Here we developed a chemogenomic screening to identify compounds that enhance senescence in PTEN-deficient cells without affecting normal cells. By using this approach, we identified casein kinase 2 (CK2) as a pro-senescent target. Mechanistically, we show that Pten loss increases CK2 levels by activating STAT3. CK2 upregulation in Pten null tumours affects the stability of Pml, an essential regulator of senescence. However, CK2 inhibition stabilizes Pml levels enhancing senescence in Pten null tumours. Taken together, our screening strategy has identified a novel STAT3-CK2-PML network that can be targeted for pro-senescence therapy for cancer

T regulatory cells are markers of disease activity in multiple sclerosis patients

FoxP3+ Treg cells are believed to play a role in the occurrence of autoimmunity and in the determination of clinical recurrences. Contradictory reports are, however, available describing frequency and function of Treg cells during autoimmune diseases. We examined, by both polychromatic flow cytometry, and real-time RT-PCR, several Treg markers in peripheral blood mononuclear cells from patients with multiple sclerosis (MS), an autoimmune disease affecting the central nervous system. We found that Tregs, as defined by CD25, CD39, FoxP3, CTLA4, and GITR expression, were significantly decreased in stable MS patients as compared to healthy donors, but, surprisingly, restored to normal levels during an acute clinical attack. We conclude that Treg cells are not involved in causing clinical relapses, but rather react to inflammation in the attempt to restore homeostasis

T Regulatory Cells Are Markers of Disease Activity in Multiple Sclerosis Patients

FoxP3+ Treg cells are believed to play a role in the occurrence of autoimmunity and in the determination of clinical recurrences. Contradictory reports are, however, available describing frequency and function of Treg cells during autoimmune diseases. We examined, by both polychromatic flow cytometry, and real-time RT-PCR, several Treg markers in peripheral blood mononuclear cells from patients with multiple sclerosis (MS), an autoimmune disease affecting the central nervous system. We found that Tregs, as defined by CD25, CD39, FoxP3, CTLA4, and GITR expression, were significantly decreased in stable MS patients as compared to healthy donors, but, surprisingly, restored to normal levels during an acute clinical attack. We conclude that Treg cells are not involved in causing clinical relapses, but rather react to inflammation in the attempt to restore homeostasis

Studio delle caratteristiche fenotipiche e funzionali di sottopopolazioni linfocitarie immunomodulatorie in pazienti affetti da sclerosi multipla

La sclerosi multipla è una patologia infiammatoria demielinizzante che colpisce il sistema nervoso centrale. Un’ipotesi generalmente condivisa è quella secondo la quale alterazioni a carico della composizione e della funzionalità dei linfociti T regolatori possano essere implicate nel processo autoimmune che contribuisce alla patogenesi della malattia. Ad oggi molteplici gruppi hanno riscontrato cambiamenti nel fenotipo e nella frequenza dei linfociti regolatori in pazienti affetti da sclerosi multipla. Sebbene negli ultimi anni il mondo scientifico abbia dedicato un notevole impegno allo studio dei linfociti T regolatori, la caratterizzazione funzionale nonché il chiarimento dei meccanismi attraverso i quali queste cellule esercitano la loro attività soppressoria non sono ancora stati ben compresi. Il presente studio si inserisce in questo ambito e nasce con lo scopo di approfondire la conoscenza dei linfociti T regolatori negli individui sani e in pazienti affetti da sclerosi multipla. Abbiamo dunque riscontrato come la popolazione CD4+CD25high caratterizzata da attività soppressoria, sia contraddistinta nell’uomo dall’espressione del CD39, un enzima appartenente alla famiglia delle nucleosidi trifosfato difosfoidrolasi, che idrolizzano i nucleotidi extracellulari nei rispettivi nucleosidi. La scissione dell’ATP da parte del CD39 avvia una reazione che si conclude con la produzione di adenosina, una molecola dalle spiccate proprietà anti-infiammatorie. L’applicazione dell’analisi citofluorimetrica a pazienti affetti da sclerosi multipla nella forma recidivante - remittente ha rivelato una diminuzione significativa della frazione regolatoria CD4+CD25highCD39+ nel sangue periferico dei pazienti monitorati. Al fine di definire eventuali correlazioni esistenti tra le caratteristiche fenotipiche e funzionali dei linfociti T regoaltori e lo stato clinico dei pazienti e allo scopo di approfondire gli effetti esercitati su questo subset cellulare dai farmaci immunomodulatori, abbiamo poi studiato un gruppo di pazienti sottoposti a trattamento con interferon β. Abbiamo misurato la frequenza della popolazione CD4+CD25highCD39+ prima del trattamento e nel corso di un anno dall’inizio di quest’ultimo. I dati ottenuti hanno messo in luce un progressivo aumento della popolazione di linfociti regolatori; tale aumento risulta visibile già dal primo time point, e viene confermato a 6 mesi dall’inizio del trattamento. Abbiamo infine applicato la nostra analisi ai linfociti effettori recentemente attivati che esprimono alti livelli di CD25 e sono in grado di rilasciare citochine pro-infiammatorie quali IFNgamma e IL17. L’azione svolta da queste popolazioni linfocitarie viene considerata critica nella patogenesi della sclerosi multipla, poiché esse partecipano attivamente al processo infiammatorio generato dalla risposta autoimmune. Abbiamo dunque riscontrato come la diminuzione della popolazione di linfociti T regolatori CD39+ sia controbilanciata, nei pazienti, da un aumento significativo delle popolazioni linfocitarie effettrici Th1 e Th17. Tali dati suggeriscono che nei pazienti affetti da sclerosi multipla l’equilibrio tra cellule effettrici e linfociti regolatori sia spostato verso la popolazione linfocitaria che svolge un’attività pro-infiammatoria.Multiple sclerosis is an inflammatory demielinating disease that affects the central nervous system. It is generally thought that subset composition and functional status of T regulatory lymphocytes could be implicated in the autoimmune process that contributes to its pathogenesis. Actually many groups have identified alterations in both the phenotype and frequency of Tregs in Multiple Sclerosis. Although in recent years an impressive amount of effort has been dedicated to the study of T regulatory cells (Tregs), the characterization of all the functional subsets as well as the elucidation of the mechanism by which these cells exert their suppressive function have not been yet accomplished. In particular for patients suffering of multiple sclerosis (MS) a significant decrease in the suppressive capacity of CD4+CD25high cells has been reported. In the present study we have performed a phenotipical and functional analysis of the T regulatory compartment in healthy controls and patients affected by the relapsing remitting form of multiple sclerosis. We have found that in humans Tregs endowed with the most potent suppressive abilities express CD39, an ectoenzyme belonging to the family of nucleoside triphosphate diphosphohydrolases (NTPDase), which hydrolyze extracellular nucleotides to the respective nucleosides. Thus, extracellular ATP can be hydrolyzed to AMP and then further to adenosine, a molecule with known anti-inflammatory properties. Interestingly, patients affected by the relapsing-remitting form of multiple sclerosis (MS) have reduced numbers of CD39+ cells within the CD4+CD25high cell population. In the attempt to define phenotypic and functional correlations with the clinical state of MS patients and to monitor the effects of immunomodulatory therapy, we have studied a cohort of MS patients undergoing treatment with β-interferon. We have measured the frequency of CD4+CD25highCD39+ cells before treatment and at 3, 6, and 9 months after the initiation of the therapy. We have found a significant increase in the fraction of CD39+ T lymphocytes already at the first time point; these data were confirmed after 6 months of therapy. We then extended our analysis to the frequency of Th1 and Th17 effector T lymphocytes, which are thought to play a fundamental role in the autoimmune process. We found that the decrease of the frequency of the CD39+ cells among the CD25high subset in these patients is reflected by an increase in the CD39- CD25high recently activated effector T cells releasing IFNγ and IL17. These data suggest that in MS patients the equilibrium between effector cells and regulatory subsets is shifted towards the proinflammatory cytokine producing cells

CD28 ligation in the absence of TCR stimulation up-regulates IL-17A and pro-inflammatory cytokines in relapsing-remitting multiple sclerosis T lymphocytes

International audienceCD28 is a crucial costimulatory receptor necessary full T cell activation. The role of CD28 in multiple sclerosis (MS) has been evaluated as the source of costimulatory signals integrating those delivered by TCR. However, CD28 is also able to act as a unique signaling receptor and to deliver TCR-independent autonomous signals, which regulate the expression and production of pro-inflammatory cytokines and chemokines. By comparing the cytokine/chemokine profiles of CD4 + T cells from relapsing-remitting multiple sclerosis (RRMS) patients and healthy donors (HD), we found that CD28 engagement without TCR strongly up-regulates IL-8 and IL-6 expression in RRMS compared to HD. More interestingly, in RRMS but not in HD, CD28 stimulation selectively induces the expression of IL-17A by cooperating with IL-6-mediated signals. By using specific inhibitory drugs, we also identify the phosphatidylinositol 3 kinase (PI3K) as the critical regulator of CD28 proinflammatory functions in MS

Reversible senescence in human CD4+CD45RA+CD27- memory T cells

Persistent viral infections and inflammatory syndromes induce the accumulation of T cells with characteristics of terminal differentiation or senescence. However, the mechanism that regulates the end-stage differentiation of these cells is unclear. Human CD4+ effector memory (EM) T cells (CD27−CD45RA−) and also EM T cells that re-express CD45RA (CD27−CD45RA+; EMRA) have many characteristics of end-stage differentiation. These include the expression of surface KLRG1 and CD57, reduced replicative capacity, decreased survival, and high expression of nuclear γH2AX after TCR activation. A paradoxical observation was that although CD4+ EMRA T cells exhibit defective telomerase activity after activation, they have significantly longer telomeres than central memory (CM)-like (CD27+CD45RA−) and EM (CD27−CD45RA−) CD4+ T cells. This suggested that telomerase activity was actively inhibited in this population. Because proinflammatory cytokines such as TNF-α inhibited telomerase activity in T cells via a p38 MAPK pathway, we investigated the involvement of p38 signaling in CD4+ EMRA T cells. We found that the expression of both total and phosphorylated p38 was highest in the EM and EMRA compared with that of other CD4+ T cell subsets. Furthermore, the inhibition of p38 signaling, especially in CD4+ EMRA T cells, significantly enhanced their telomerase activity and survival after TCR activation. Thus, activation of the p38 MAPK pathway is directly involved in certain senescence characteristics of highly differentiated CD4+ T cells. In particular, CD4+ EMRA T cells have features of telomere-independent senescence that are regulated by active cell signaling pathways that are reversible

Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4(+) CD45RA(+) CD27(-) T cells: the potential involvement of interleukin-7 in this process

P>The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4+ T-cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA+ CD27+ CD4+ T cells over time. In contrast, the increase in CD45RA- CD27- and CD45RA+ CD27- CD4+ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RA- CD27- and CD45RA+ CD27- CD4+ T cells in CMV-seropositive donors are specific for this virus. CD45RA+ CD27- CD4+ T cells have significantly reduced CD28, interleukin-7 receptor alpha (IL-7R alpha) and Bcl-2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T-cell receptor activation compared with the other T-cell subsets in the same donors. Despite this, the CD45RA+ CD27- subset is as multifunctional as the CD45RA- CD27+ and CD45RA- CD27- CD4+ T-cell subsets, indicating that they are not an exhausted population. In addition, CD45RA+ CD27- CD4+ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4+ memory T cells re-expressing CD45RA can be generated from the CD45RA- CD27+ population by the addition of IL-7 and during this process these cells down-regulated expression of IL-7R and Bcl-2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA+ CD27- CD4+ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated