Alessia Zoso

Ubiquitin-Proteasome System Stress Sensitizes Ovarian Cancer to Proteasome Inhibitor–Induced Apoptosis

The ubiquitin-proteasome system (UPS) mediates targeted protein degradation. Notably, the UPS determines levels of key checkpoint proteins controlling apoptosis and proliferation by controlling protein half-life. Herein, we show that ovarian carcinoma manifests an overstressed UPS by comparison with normal tissues by accumulation of ubiquitinated proteins despite elevated proteasome levels. Elevated levels of total ubiquitinated proteins and 19S and 20S proteasome subunits are evident in both low-grade and high-grade ovarian carcinoma tissues relative to benign ovarian tumors and in ovarian carcinoma cell lines relative to immortalized surface epithelium. We find that ovarian carcinoma cell lines exhibit greater sensitivity to apoptosis in response to proteasome inhibitors than immortalized ovarian surface epithelial cells. This sensitivity correlates with increased cellular proliferation rate and UPS stress rather than absolute proteasome levels. Proteasomal inhibition in vitro induces cell cycle arrest and the accumulation of p21 and p27 and triggers apoptosis via activation of caspase-3. Furthermore, treatment with the licensed proteasome inhibitor PS-341 slows the growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice. In sum, elevated proliferation and metabolic rate resulting from malignant transformation of the epithelium stresses the UPS and renders ovarian carcinoma more sensitive to apoptosis in response to proteasomal inhibition.

Aptamer-Mediated Blockade of IL4Rα Triggers Apoptosis of MDSCs and Limits Tumor Progression

In addition to promoting tumor progression and metastasis by enhancing angiogenesis and invasion, myeloid-derived suppressor cells (MDSC) and tumor-associated macrophage (TAM) also inhibit antitumor T-cell functions and limit the efficacy of immunotherapeutic interventions. Despite the importance of these leukocyte populations, a simple method for their specific depletion has not been developed. In this study, we generated an RNA aptamer that blocks the murine or human IL-4 receptor-α (IL4Rα or CD124) that is critical for MDSC suppression function. In tumor-bearing mice, this anti-IL4Rα aptamer preferentially targeted MDSCs and TAM and unexpectedly promoted their elimination, an effect that was associated with an increased number of tumor-infiltrating T cells and a reduction in tumor growth. Mechanistic investigations of aptamer-triggered apoptosis in MDSCs confirmed the importance of IL4Ra-STAT6 pathway activation in MDSC survival. Our findings define a straightforward strategy to deplete MDSCs and TAMs in vivo, and they strengthen the concept that IL4Rα signaling is pivotal for MDSC survival. More broadly, these findings suggest therapeutic strategies based on IL4Rα signaling blockades to arrest an important cellular mechanism of tumoral immune escape mediated by MDSCs and TAM in cancer.

In vivo induction of myeloid suppressor cells and CD4(+)Foxp3(+) T regulatory cells prolongs skin allograft survival in mice.

Natural CD4+Foxp3+ T regulatory (Treg) cells can promote transplantation acceptance across major histocompatibility complex (MHC) barriers, while myeloid-derived suppressor cells (MDSCs) inhibit effector T-cell responses in tumor-bearing mice. One outstanding issue is whether combining the potent suppressive function of MDSCs with that of Treg cells might synergistically favor graft tolerance. In the present study, we evaluated the therapeutic potential of MDSCs and natural Treg cells in promoting allograft tolerance in mice by utilizing immunomodulatory agents to expand these cells in vivo. Upon administration of recombinant human granulocyte-colony stimulating factor (G-CSF; Neupogen), or interleukin-2 complex (IL-2C), Gr-1+CD11b+ MDSCs or CD4+Foxp3+ Treg cells were respectively induced at a high frequency in the peripheral lymphoid compartments of treated mice. Interestingly, induced MDSCs exhibited a more potent suppressive function in vitro when compared to MDSCs from naive mice. Furthermore, in vivo coadministration of Neupogen and IL-2C induced MDSCs at percentages that were higher than those seen when either agent was administered alone, suggesting an additive effect of the two drugs. Although treatment with either IL-2C or Neupogen led to a significant delay of MHC class II disparate allogeneic donor skin rejection, the combinatorial treatment was superior to either alone. Importantly, histological assessment of surviving grafts revealed intact morphology and minimal infiltrates at 60 days posttransplant. Collectively, our findings demonstrate that concurrent induction of MDSCs and Tregs is efficacious in downmodulating alloreactive T-cell responses in a synergistic manner and highlight the therapeutic potential of these naturally occurring suppressive leukocytes to promote transplantation tolerance.

In vivo Administration of Artificial Antigen-Presenting Cells Activates Low-Avidity T Cells for Treatment of Cancer

The development of effective antitumor immune responses is normally constrained by low-avidity, tumor-specific CTLs that are unable to eradicate the tumor. Strategies to rescue antitumor activity of low-avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low-avidity CTLs, we immunized mice bearing lung melanoma metastases with artificial antigen-presenting cells (aAPC), made by covalently coupling (pep)MHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system, and complete tumor eradication in a mouse TRP-2 antigen system, when low-avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis, and TCR downregulation in low-avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherapy.

Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion

By restraining T‐cell activation and promoting Treg‐cell expansion, myeloid‐derived suppressor cells (MDSCs) and tolerogenic DCs can control self‐reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor‐free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4‐day culture with FDA‐approved cytokines (recombinant human‐GM‐CSF and recombinant human‐G‐CSF). This MDSC subset, characterized by the expression of MDSC‐, DC‐, and fibrocyte‐associated markers, promotes Treg‐cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.

Moderate Intensity Training Impact on the Inflammatory Status and Glycemic Profiles in NOD Mice

The nonobese diabetic (NOD) mouse represents a well-established experimental model analogous to human type 1 diabetes mellitus (T1D) as it is characterized by progressive autoimmune destruction of pancreatic β-cells. Experiments were designed to investigate the impact of moderate-intensity training on T1D immunomodulation and inflammation. Under a chronic exercise regime, NOD mice were trained on a treadmill for 12 weeks (12 m/min for 30 min, 5 d/wk) while age-matched, control animals were left untrained. Prior to and upon completion of the training period, fed plasma glucose and immunological soluble factors were monitored. Both groups showed deteriorated glycemic profiles throughout the study although trained mice tended to be more compensated than controls after 10 weeks of training. An exercise-induced weight loss was detected in the trained mice with respect to the controls from week 6. After 12 weeks, IL-6 and MIP-1β were decreased in the trained animals compared to their baseline values and versus controls, although not significantly. Morphometric analysis of pancreata revealed the presence of larger infiltrates along with decreased α-cells areas in the control mice compared to trained mice. Exercise may exert positive immunomodulation of systemic functions with respect to both T1D and inflammation, but only in a stringent therapeutic window.

Gene expression profiling of human fibrocytic myeloid-derived suppressor cells (f-MDSCs)

Myeloid-derived suppressor cells (MDSCs) have been shown to control self-reactive and anti-graft effector T-cells in autoimmunity and transplantation, but their therapeutic use is limited by their scarce availability in the peripheral blood of tumor-free donors. We isolated and characterized a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC), which are differentiated from umbilical cord blood (UCB) precursors (Zoso et al., 2014). This MDSC subset promotes regulatory T-cell expansion and induces normoglycemia in a xenogeneic model of type 1 diabetes. Here we describe in details the experimental design and the bioinformatics analyses of the gene expression dataset used to investigate the molecular mechanisms at the base of MDSC tolerogenic and suppressive properties. We also provide an R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset. Raw and pre-processed data are available at Gene Expression Omnibus under accession GSE52376.

MHC-Ig induces memory T cell formation in vivo and inhibits tumour growth.

Induction of a T cell mediated immune response is critical for the eradication of viral infections and tumours. Soluble peptide‐loaded major histocompatibility complex‐Ig (pep−MHC‐Ig) have been shown to bind their cognate ligands, T cell receptor, with high affinity, and are successfully used to visualize antigen‐specific T cells. Furthermore, immobilized pep−MHC‐Ig can activate and expand antigen‐specific T cells in vitro and in vivo. In this study, we investigate the use of pep−MHC‐Ig as a potential strategy to modulate antigen specific T cell immune responses in vivo. SIY−Kb‐Ig immunization, together with the pre‐activation by an anti‐CD40 monoclonal antibody, is able to stimulate a strong expansion of adoptively transferred 2C transgenic T cells and the formation of long term antigen‐specific memory T cells. In addition, mechanistic studies show that the pep−MHC‐Ig molecules directly activate T cells in vivo without requiring uptake and reprocessing by antigen‐presenting cells. Furthermore, B6 mice immunized with pep−MHC‐Ig molecules inhibit tumour growth in a B16‐SIY melanoma prevention model. Thus, soluble pep−MHC‐Ig molecules represent a powerful tool for active immunotherapy.

G-CSF and Exenatide Might Be Associated with Increased Long-Term Survival of Allogeneic Pancreatic Islet Grafts

Background Allogeneic human islet transplantation is an effective therapy for the treatment of patients with Type 1 Diabetes (T1D). The low number of islet transplants performed worldwide and the different transplantation protocols used limit the identification of the most effective therapeutic options to improve the efficacy of this approach. Methods We present a retrospective analysis on the data collected from 44 patients with T1D who underwent islet transplantation at our institute between 2000 and 2007. Several variables were included: recipient demographics and immunological characteristics, donor and transplant characteristics, induction protocols, and additional medical treatment received. Immunosuppression was induced with anti-CD25 (Daclizumab), alone or in association with anti-tumor necrosis factor alpha (TNF-α) treatments (Etanercept or Infliximab), or with anti-CD52 (Alemtuzumab) in association with anti-TNF-α treatments (Etanercept or Infliximab). Subsets of patients were treated with Filgrastim for moderate/severe neutropenia and/or Exenatide for post prandial hyperglycemia. Results The analysis performed indicates a negative association between graft survival (c-peptide level ≥ 0.3 ng/ml) and islet infusion volume, with the caveat that, the progressive reduction of infusion volumes over the years has been paralleled by improved immunosuppressive protocols. A positive association is instead suggested between graft survival and administration of Exenatide and Filgrastim, alone or in combination. Conclusion This retrospective analysis may be of assistance to further improve long-term outcomes of protocols for transplant of islets and other organs.