GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo

We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC

Apoptotic epitope-specific CD8+ T cells and interferon signaling intersect in chronic hepatitis C virus infection

CD8(+) T cells specific to caspase-cleaved antigens derived from apoptotic T cells represent a principal player in chronic immune activation (CIA). Here, we found that both apoptotic epitope (AE)-specific and hepatitis C virus (HCV)-specific CD8(+) T cells were mostly confined within the effector memory (EM) or terminally differentiated EM CD45RA(+) cell subsets expressing a dysfunctional T-helper-1-like signature program in chronic (c)HCV infection. However, AE-specific CD8(+) T cells produced tumor necrosis factor (TNF)-α and interleukin-2 at the intrahepatic level significantly more than HCV-specific CD8(+) T cells, despite both populations acquiring high levels of programmed death-1 receptor expression. Contextually, only AE-specific CD8(+) T cells correlated with both interferon-stimulated gene levels in T cells and hepatic fibrosis score. Taken together, these data suggest that AE-specific CD8(+) T cells can sustain CIA by their capacity to produce TNF-α and be resistant to inhibitory signals more than HCV-specific CD8(+) T cells in cHCV infection

Expansion of activated regulatory T cells inversely correlates with clinical severity in septic neonates.

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Combination of chemotherapy and PD-1 blockade induces T cell responses to tumor non-mutated neoantigens

Here, we developed an unbiased, functional target-discovery platform to identify immunogenic proteins from primary non-small cell lung cancer (NSCLC) cells that had been induced to apoptosis by cisplatin (CDDP) treatment in vitro, as compared with their live counterparts. Among the multitude of proteins identified, some of them were represented as fragmented proteins in apoptotic tumor cells, and acted as non-mutated neoantigens (NM-neoAgs). Indeed, only the fragmented proteins elicited effective multi-specific CD4+ and CD8+ T cell responses, upon a chemotherapy protocol including CDDP. Importantly, these responses further increased upon anti-PD-1 therapy, and correlated with patients’ survival and decreased PD-1 expression. Cross-presentation assays showed that NM-neoAgs were unveiled in apoptotic tumor cells as the result of caspase-dependent proteolytic activity of cellular proteins. Our study demonstrates that apoptotic tumor cells generate a repertoire of immunogenic NM-neoAgs that could be potentially used for developing effective T cell-based immunotherapy across multiple cancer patients

CD8+ T cells specific to apoptosis-associated epitopes are expanded in patients with chronic HBV infection and fibrosis

BACKGROUND & AIMS: During chronic viral infections, the apoptosis of activated T cell elicits a CD8+ T cell response directed to those cryptic epitopes that emerge from caspase-cleaved structural proteins. Such response directed to apoptosis-associated epitopes (AE) contributes to the amplification of immunopathology.METHODS: Here, we have analysed through flow cytometry AE-specific CD8+ T cells in patients with chronic hepatitis B virus (HBV) infection, naive-to-treatment or undergoing nucleos(t)ide-analogue (NUC) therapy.RESULTS: We found that AE-specific CD8+ T cell frequencies were significantly increased only in those NUC-treated patients who also presented advanced hepatic fibrosis. Regulatory T cells were also expanded in those patients, and AE-specific, but not HBV-specific, CD8+ T cell frequency positively correlated with Treg percentages. Through multiparameter flow cytometry, multidimensionality reduction and unsupervised clustering analysis, we could identify novel subpopulations among effector memory (em) and emCD45RA+ T cell (Tem and Temra) subsets. CD8+ T cells with distinct specificities differentially populated the subpopulation map: while HBV-specific were mostly contained in the Tem subset, AE-specific CD8+ T cells encompassed naive, as well as T central memory, Tem and Temra cells.CONCLUSION: All together, these findings indicate a link between AE-specific CD8+ T cells and advanced liver fibrosis in patients with chronic HBV infection, and suggest that virus-specific and AE-specific CD8+ T cells exhibit distinct differentiation states and contribute in distinct ways to immunopathology

CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis.

CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes) represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control). The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients

Biological activity of 3-chloro-azetidin-2-one derivatives having interesting antiproliferative activity on human breast cancer cell lines

Resveratrol (3,40,5 tri-hydroxystilbene), a natural plant polyphenol, has gained interest as a non-toxic agent capable of inducing tumor cell death in a variety of cancer types. However, therapeutic application of these beneficial effects remains very limited due to its short biological half-life, labile properties, rapid metabolism and elimination. Different studies were undertaken to obtain synthetic analogs of resveratrol with major bioavailability and anticancer activity. We have synthesized a series 3-chloro-azetidin-2-one derivatives, in which an azetidinone nucleus connects two aromatic rings. Aim of the present study was to investigate the effects of these new 3-chloro-azetidin-2-one resveratrol derivatives on human breast cancer cell lines proliferation. Our results indicate that some azetidin-based resveratrol derivatives may become new potent alternative tools for the treatment of human breast cance

IFN-α promotes rapid human Treg contraction and late Th1-like Treg decrease

Type I IFNs are pleiotropic cytokines that exert concerted activities in the development of antiviral responses. Regulatory T cells represent a physiologic checkpoint in the balance between immunity and tolerance, requiring fine and rapid controls. Here, we show that human regulatory T cells are particularly sensitive to the sequential effects of IFN-α. First, IFN-α exerts a rapid, antiproliferative and proapoptotic effect in vitro and in vivo, as early as after 2 d of pegylated IFN/ribavirin therapy in patients with chronic hepatitis C. Such activities result in the decline, at d 2, in circulating regulatory T cell frequency and specifically of the activated regulatory T cell subset. Later, IFN-based therapy restrains the fraction of regulatory T cells that can be polarized into IFN-γ-producing Th1-like regulatory T cells known to contribute to chronic immune activation in type 1 inflammation. Indeed, Th1-like regulatory T cell frequency significantly declines after 30 d of therapy in vivo in relation to the persistent decline of relevant IL-12 sources, namely, myeloid and 6-sulfo LacNAc-expressing dendritic cells. This event is recapitulated by experiments in vitro, providing evidence that it may be attributable to the inhibitory effect of IFN-α on IL-12-induced, Th1-like regulatory T cell polarization. In summary, our results suggest that IFN-α-driven, early regulatory T cell depletion contributes to the development of antiviral immunity, ultimately resulting in the resolution of type 1 inflammation

AE-specific dextramer⁺CD8⁺ T cells specifically producing inflammatory cytokines inversely correlate with PD-1⁺dextramer⁺CD8⁺ T cells (A-C).

<p>(A) Representative flow cytometry analyses of cells producing IL-17 or IFN-<b>γ</b> in dextramer⁺CD8⁺ T cells in response to a relevant AE pool (5 peptides). (B) Percentage of cytokine-producing cells in dextramer⁺CD8⁺ T cells from Rs and NRs. Statistical analysis was performed with the Mann-Whitney test. ns = not significant. (C) Correlation between cytokine-producing dextramer⁺CD8⁺ T cells and those expressing PD-1 (Spearman correlation analysis). Analyses were performed at time 0 (before the start of therapy).</p

CD8⁺ T cell multispecificity to AEs in HDs and RA patients (A-C).

<p>(A) Mean number of IFN-<b>γ</b> spots formed by fresh CD8⁺ T_EM cells (by ELISPOT assay) in response to 12 pools of AEs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128607#pone.0128607.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128607#pone.0128607.s004" target="_blank">S4</a> Tables) in 12 HLA-A2⁺ patients with RA or 24 HLA-A2⁺ HDs. (B) Sum of IFN-<b>γ</b> spots formed by fresh CD8⁺ TEM cells in response to all pools (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128607#pone.0128607.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128607#pone.0128607.s004" target="_blank">S4</a> Tables) of AEs in the single patient or HD. (C) Sum of IFN-<b>γ</b> spots formed by fresh CD8⁺ TEM cells in response to all pools (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128607#pone.0128607.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128607#pone.0128607.s004" target="_blank">S4</a> Tables) of AEs in the single R, NR, or HD. Analyses were performed at time 0 (before the start of therapy). Statistical analysis was performed with the Mann-Whitney test. *<i>P</i> < 0.01; **<i>P</i> < 0.001; ***<i>P</i> < 0.0001. ns = not significant. RLA2 = 60S acidic ribosomal protein P2; PSA1 = proteasome component C2; VIME = vimentin; GDIS = rho GDP dissociation inhibitor 2; Myh9 = non-muscle myosin; LAM1 = Iamin B1; ROK = heterogeneous nuclear ribonucleoprotein K; ACT B = actin cytoplasmic 1.</p