extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry Medicine

text:
Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

AIM To investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis. METHODS The expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed. RESULTS The expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs. CONCLUSION ELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling.

INTRODUCTION
Liver fibrosis occurs in most chronic liver diseases and is characterized by the accumulation of extracellular matrix following sustained inflammation [1] . The main causes of liver fibrosis include chronic HBV and HCV infection, parasitic infections, alcohol abuse, and NASH. Liver fibrosis can lead to the dysfunction of hepatocytes and obstruction of the intrahepatic blood flow, which resulting in the subsequent hepatic insufficiency and hypertension of the portal vein [2] . HSCs, which are the main source of ECM in injured livers, play a crucial role in the progress of liver fibrosis. In normal livers, HSCs are located in the Disse space, between the sinusoidal endothelium and hepatocytes. HSCs become activated and acquire contractile, proinflammatory, and fibrogenic properties when subjected to diverse chronic injuries. Researchers have identified many factors that promote the transdifferentiation of quiescent HSCs into activated HSCs [3] .
The TGF-β signaling plays a very important role in various cellular physiological processes through the regulation of downstream proteins [4] . Frequent inactivation of the TGF-β pathway components in liver fibrosis demonstrates a powerful promoting role of the TGF-β pathway. The multifunctional effects of TGF-β in cellular actions occur through the binding of its receptors, TGF-β receptor II and receptor I; the activation of intrinsic kinase activity; and the phosphorylation and translocation of mediators, Smads, followed by TGF-β target gene activation [5] . ELF, also known as β2-spectrin, is important for maintaining function of cellular membranes and polarization of epithelial cells. Meanwhile, ELF also plays an important role in TGF-β pathway.
Smad 3 and Smad 4 are the main proteins of Smad family which is crucial for the activation of TGF-β pathway. The interaction of ELF and Smad proteins was able to facilitate the transport of Smad 3-Smad4 complex into nucleus, which lead to the subsequent activation of TGF-β pathway [6][7][8] .
Our previous study demonstrated that ELF is involved in the activation of HSCs. First, Western blot and RT-qPCR evaluation indicated that ELF expression was increased remarkably in fibrotic mouse model. Moreover, the silence of ELF in activated HSCs significantly reduced the ECM components such as collagen and α-SMA. Clarification of the mechanism is needed [9] . In addition, our study showed that the PI3K/Akt signaling is regulated by ELF in the process of hepatocyte proliferation [10] . The PI3K/Akt signaling is essential in the regulation of glucose metabolism in many types of cells [11,12] . Researchers have identified a novel mechanism for reprogramming quiescent HSCs into activated HSCs that depends on the induction of glycolysis, similar to the Warburg state that has been described in cancer cells [13] . We hypothesized that ELF is involved in glucose metabolism of HSCs through PI3K/Akt signaling.

Ethics statement
All the processes were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology, Wuhan, China.

Mouse model of liver fibrosis
Eight-week-old male mice(C57/Black/6) that purchased from Animal Center of HUST (Wuhan, China) were challenged by classic subcutaneous injection with carbon tetrachloride (CCl4) diluted at a 3:7 (v/v) ratio in 5 mL/kg olive oil twice weekly for 12 wk as described previously.

HSC isolation
Primary hepatic stellate cells used in this study were isolated from C57/Black/6 mice (including control and fibrosis model) by in situ perfusion and centrifugation.
The details were described in our previous study [9] .

Preparation and transfection of siRNAs
siRNA used to silence ELF expression was synthesized by Applied Biosystems. The concentration of siRNAs (and scrambled siRNAs) was 50 pmol/L. Lipo2000 (Invitrogen) was used to transduce the siRNA into mice hepatic stellate cells on six wells when the confluence was about 30%-50%. RNA extraction was performed 72 h later. According to previous identification, ELF siRNA s74307 was chosen because of its best efficacy.

Western blot
The total protein was extracted from cell and tissue using RIPA buffer with protease inhibitors. Concentrations of proteins were evaluated by BCA Assay Kit. Total proteins (50 μg) were separated on 10% SDS-PAGE. The immunoblotting was performed. The immune complex was visualized by ECL detection Immunohistochemistry Liver specimens for histology and immunohistochemistry were fixed in 10% buffered formalin for 48 h, and then sliced into sections. Staining was performed using ABC kit. Sections were incubated at 4 ℃ with antibody for 12 h. DAB was used to visualize immunocomplexes.

Measurement of lactate
Whole cell lysates of liver sample and HSCs were prepared with pyruvate assay buffer and then filtered through a 10-kilodalton molecular weight spin filter for deproteinization. Levels of lactate were measured using a lactate assay kit or pyruvate assay kit from BioVision according to the manufacturer's instructions, and normalized to the control group.

Statistical analysis
The results were presented as the mean ± SD. The differences between groups were tested by Student two-tailed t test, and P < 0.05 was considered statistically significant.

ELF expression is upregulated in fibrotic liver and HSCs
To evaluate whether ELF is involved in liver fibrosis, we generated a fibrotic mouse model. The immunohistochemical (IHC) examination found that ELF expression was increased in the fibrotic livers compared with controls ( Figure 1A). Moreover, the ELF expression was observed near the bridging fibrotic areas. In addition, ELF mRNA (with RT-qPCR, approximately 2.5 times) and protein expression by Western blot analysis was increased in the CCl4-treated animals ( Figure 1B). HSCs are predominantly located in the areas of bridging fibrosis, and we isolated HSCs from the fibrotic and normal livers. The RT-qPCR and Western blot test of the ELF expression found that there was a more significant increase in the HSCs isolated from the fibrotic livers than from the normal livers ( Figure 1C). This finding indicated that the upregulated expression of ELF in Real-time RT-PCR and Western blot analysis were used to evaluate the ELF expression in the liver homogenates from the control and CCl4-treated mice. a P < 0.05, the CCl4-treated mice vs the control mice. GAPDH was used as the control; C: Real-time RT-PCR and Western blot analysis were used to evaluate the ELF expression in the primary hepatic stellate cells (HSCs) isolated from the control and CCl -treated mice. b P < 0.01, the CCl4-treated mice vs the control mice; D: The α-SMA and collagen I expression at the protein level were upregulated in the whole liver homogenates from the CCl4-treated mice compared with the controls. levels. Glucose transporter 1 (Glut 1) is a uniporter protein that in humans is encoded by the SLC2A1 gene. Glut 1 is the first glucose transporter which facilitates the transport of glucose from blood into membrane in various kinds of cells [17,18] . We found that Glut1 expression also showed a remarkable increase in fibrotic liver than control mice ( Figure 2E and F).
Monocarboxylate transporter 4 (MCT4), the lactate export pump, plays an crucial role in the accumulation of intracellular lactate in various cells [19] . The upregulation of MCT4 in fibrotic livers indicates that lactate accumulation was increased in fibrotic livers compared with the controls ( Figure 2G and H). These findings indicated that glycolysis related genes were overexpressed in fibrotic livers. However, the expression of these genes in HSCs remains unknown.
The intracellular lactate was evaluated by a lactate assay kit. As shown in Figure 2I, intracellular lactate level increased significantly in fibrotic liver.

Glycolysis related genes are upregulated significantly in HSCs isolated from fibrotic livers
The liver is composed of many cell types, and further HSCs might play an important role in liver fibrosis.

Glycolysis-related genes are upregulated in fibrotic livers
Previous study had demonstrated that glucose metabolism was reprogrammed in fibrotic livers [13] .
To confirm whether glycolysis-related genes were changed in fibrotic livers, we selected some key proteins which are involved in glucose glycolysis, such as phosphofructokinase (PFKP), Glut1, PKM2, and MCT4. PFKP, a gene which encodes the platelet isoform of phosphofructokinase (PFK), plays an important role in glucose glycolysis of various cell types [14,15] . As shown in Figure 2A and 2B, the PFKP expression was increased at both the mRNA and protein levels. Pyruvate kinase isozymes M1/M2 (PKM1/M2) is an enzyme encoded by the PKM2 gene. The function of PKM2 is to catalyze the last step within glycolysis, and leads to the dephosphorylation of phosphoenolpyruvate [16] . Realtime quantitative PCR and Western blot ( Figure 2C and D) demonstrated that PKM expression was increased significantly in fibrotic liver at both mRNA and protein The PFKP and PFKM2 expression of the mRNA level was increased compared with that of the control ( a P < 0.05 vs control). GAPDH was used as the control; B, D: Western blot analysis indicated that the PFKP and PFKM2 expression of the protein level was upregulated in the fibrotic livers; E: The real-time RT-PCR analysis evaluated the hepatic glucose transporter, Glut 1, expression in the fibrotic livers and the control mice. The expression of Glut 1 mRNA was increased compared with that of the controls ( a P < 0.05 vs control); F: Western blot analysis indicated that the expression of the Glut 1 at protein level was upregulated in the fibrotic livers; G: Real-time RT-PCR analysis evaluated the MCT4 hepatic lactate transporter expression in the fibrotic livers and the control mice. The expression of the Glut 1 mRNA was increased compared with that of the controls ( a P < 0.05 vs control); H: Western blot analysis indicated that the expression of MCT4 at protein level was upregulated in the fibrotic liver; I: The intracellular lactate was evaluated by a lactate assay kit ( a P < 0.05 vs control). H studies should be performed if a certain protein is overexpressed in the liver. Therefore, we first isolated the HSCs from a fibrotic liver by in situ perfusion. Then, the total protein and mRNA were extracted. As demonstrated in Figure 3, the expression of PFKP, GLUT 1, PKM2, and MCT4 increased remarkably (P < 0.01) shown by real time-quantitative PCR ( Figure 3A, C, E and G) and Western blot ( Figure 3B, D, F and H). Moreover, lactate ( Figure 3I) showed a significant increase compared with the controls (P < 0.01). This finding indicates that the glucose metabolism of activated HSCs was reprogrammed. The reprogrammed metabolism might supply energy to HSCs for their proliferation, ECM production, and migration.

Silence of ELF in activated HSCs leads to the inhibition of glycolysis
The efficiency of the knockdown of ELF in activated HSCs by siRNA was tested by Western blot ( Figure  4A) and real-time quantitative PCR ( Figure 4B). The ELF-siRNA treatment led to a remarkable decrease in the ELF expression. A significant reduction in SMA and collagen 1 expression was observed in the ELF-siRNA treated HSCs ( Figure 4H and I). In addition, PFKP, GLUT 1, PKM2, and MCT4 showed a significant decrease in response to the ELF-siRNA ( Figure 4C-F). The intracellular lactate also decreased significantly in response to the silence of ELF ( Figure 4G). This finding indicates that the silence of ELF leads to the inhibition of glucose glycolysis-related genes. These results indicated that silence of ELF could block HSC activation, reduce ECM production, and inhibit glycolysis.

ELF-PI3K/Akt-glycolysis axis in HSC
Although previous data indicated that ELF might play an important role in HSC activation and glycolysis in the pathogenesis of liver fibrosis, the mechanism needs to be clarified. Since PI3K/Akt signaling is important in glucose glycolysis of many cell types, and based on our previous study of the interaction between ELF and PI3K/Akt pathway, we hypothesized that the cross talk of ELF and PI3K/Akt signaling was involved in the activation of HSCs.  H To confirm this finding, we first evaluated Akt, the key protein of PI3K signaling, in ELF-siRNA treated HSCs. As shown in Figure 5A, the expression of pAkt decreased significantly after the silencing of ELF in activated HSCs, however the total AKT was not influenced by ELF-siRNA treatment. Then, we administered activated HSCs with LY294002, a PI3K/Akt signaling inhibitor, and made the following evaluation. The remarkable downregulation of the pAkt expression confirmed that the PI3K/Akt signaling was well blockaded by LY294002 ( Figure 5D). According to the Western blot and RT-qPCR testing, we found that inhibition of the PI3K/akt signaling does not affect the ELF expression ( Figure 5B and C). Therefore, the key proteins of glycolysis, PFKP and PKM2, showed a significant decrease in the activated HSCs treated with LY294002 at the mRNA ( Figure 5E and G) and protein levels ( Figure 5F and H). In addition, SMA and collagen, the main components of the extracellular matrix, were effectively repressed by the LY294002 treatment ( Figure 5I and J). Based on these data, we first demonstrated that ELF could regulate pAkt and glycolysis. Moreover, the blockade of PI3K/akt led to the repression of glycolysis and ECM production. The ELF was involved in the HSC glycolysis through the regulation of PI3K/Akt signaling.

DISCUSSION
The destruction of the sinusoidal architecture in hepatic fibrosis always leads to hypoxia of the HSCs around the sinusoids. Simultaneously, the HSCs show a strong ability to activate and proliferate, which requires sufficient energy. The mechanism by which the HSCs ensure sufficient energy for activation and proliferation in the anoxic microenvironment is unknown. As shown in recent research, glycolysis was increased significantly in activated HSCs. Cellular metabolism might be a novel aspect in the pathogenesis study of liver fibrosis. To date, most studies of liver fibrosis Figure 4 Silencing of Embryonic liver fordin in the activated hepatic stellate cells led to the inhibition of glycolysis. A: The ELF mRNA was reduced in the activated hepatic stellate cells (HSCs) transfected synthetic siRNA against ELF was assessed by real-time RT-PCR. P < 0.01 for the ELF siRNA versus the siRNA controls. GAPDH was used as the control; B: Western blot analysis confirmed that synthetic siRNA inhibited the ELF expression in the activated HSCs; C, D: The hepatic glycolytic enzymes, PFKP and PFKM2, expression of the mRNA and protein level decreased significantly after the ELF siRNA treatment ( a P < 0.05 vs control). E, F: The hepatic glycolytic enzymes, Glut 1 and MCT 4, expression of the mRNA at the protein level decreased significantly after the ELF siRNA treatment ( a P < 0.05 vs control). G: The intracellular lactate level decreased significantly after the ELF siRNA treatment ( a P < 0.05 vs control); H, I: The main components of the extracellular matrix, α-SMA and collagen I, expression showed a remarkable decrease in the activated HSCs, and the HSC transfected synthetic siRNA against ELF. focused on HSC activation and ECM production. We showed that energy metabolism, particularly glycolysis, represents an innovative role in HSC activation.
In this study, we found a novel role for ELF in the metabolic process of HSC s activation; the PI3K/Akt pathway was shown to regulate the glycolysis of HSCs. We first found that ELF expression was increased in the HSCs of the fibrosis mouse model and the HSCs cultured for 3 wk in vitro. PFKP is the key enzyme in glycolysis and GLU, which showed a significant increase in the activated HSCs compared with the controls. Next, we tested whether increased ELF expression affects glycolysis related-proteins. The silencing of ELF in the activated HSCs leads to decreased expression of glycolysis-related enzymes, such as PFKP, GLUT1, PKM2, and MCT4. Further studies indicated that ELF was involved in HSC glucose metabolism through regulation of pAkt, an important downstream factor in PI3K/Akt signaling.
PI3K signaling, one of the most important pathways that involved various human diseases, provides intense growth and survival signals to many cell types and has profound effects on cellular metabolism [20] . As demonstrated in previous studies, the integration of growth and proliferation signals with alterations to the central metabolism is important for the oncogenic effects of the PI3K pathway [21,22] . Protein kinase B (PKB), also known as Akt, is a serine/ threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. Akt is the best-studied downstream factor of PI3K signaling, and an important driver of the glycolytic phenotype tumor, and stimulates ATP generation, ensuring that cells have the bioenergetic capacity required to respond to growth signals [23] . Akt was able to phosphorylate key glycolytic enzymes such as hexokinase, phosphofructokinase 2, and Glu 1. This function leads to increased expression and membrane translocation of the glucose transporters [24][25][26] . Abundant evidence has demonstrated that PI3K/Akt pathway is crucial for the proliferation and metabolism of many kinds of cells. Therefore, many studies have suggested that TGF-β is able to activate PI3K/Akt signaling and leads to the subsequent phosphorylation of the downstream genes without an interaction with Smad proteins [27][28][29] . TGF-β was shown to downregulate PI3K/Akt signaling activity The key protein of PI3K/akt signaling, pAKT, decreased significantly in the embryonic liver fordin (ELF)-siRNA treated hepatic stellate cells (HSCs), but the expression of total AKT was not affected by ELF-siRNA treatment. GAPDH was used as the control; B, C: The Western blot and real-time RT-PCR analysis indicated that the ELF expression was not affected by Ly294002, an inhibitor of the PI3K/akt signaling; D: The pAKT expression decreased obviously after the inhibition of the PI3K/akt signaling in the HSCs; E, F: The hepatic glycolytic enzymes PFKP expression of mRNA and the protein level decreased significantly after the Ly294002 treatment in the activated HSCs ( a P < 0.05 vs control); G, H: The hepatic glycolytic enzyme PKM2 expression at mRNA and the protein levels decreased significantly after the Ly294002 treatment in the activated HSCs ( a P < 0.05 vs control). I, J: The main components of the extracellular matrix, α-SMA and collagen I expression showed a remarkable decrease in the activated HSCs treated by the PI3K/akt signaling inhibitor, Ly294002.  [30,31] . Therefore, the PI3K/Akt signaling can also antagonize the Smad-mediated effects in other TGF-β related conditions [32,33] . Our previous studies showed that the activation of PI3K/ Akt signaling through insulin stimulation induced the activation of TGF-β/Smad signaling, as indicated by the nuclear translocation of Smad3/4. Moreover, the inhibition of PI3K/Akt signaling by the use of the LY294002 inhibitor led to the blockage of TGF-β/Smad signaling [9] . These findings indicated that the activated PI3k/Akt pathway led to the activation of TGF-β/Smad signaling in the hepatocytes. ELF was shown to play an important role in this process by regulating the localization of Smad3/4 in the nucleus [10] . However, the details underlying the interaction between TGF-β/ Smad and PI3K/Akt signaling, particularly in the field of cell energy metabolism, should be investigated in the future.
Multiple intrinsic and extrinsic molecular mechanisms contribute to cellular metabolism and provide support for the three basic needs of dividing cells, as follows: rapid ATP generation to maintain the energy status; increased biosynthesis of macromolecules; and tightened maintenance of appropriate cellular redox status [34,35] . Metabolic changes represent a common feature of all cell types and various diseases including liver fibrosis. This effect is regulated by diverse pathways and factors, such as PI3K signaling, the hypoxia-inducible factor, p53, MYC and AMPactivated protein kinase, liver kinase B1, and the hedgehog pathways [36,37] . Liver fibrosis represents a very complicated process. In recent years, scientists have revealed various pathways that contribute to the pathogenesis of liver fibrosis. Although we showed that PI3K/Akt, which was regulated by ELF, could affect glycolysis in HSCs, other signaling could perform this function. Additional signals that were involved in the regulation of energy metabolism should be investigated in liver fibrosis. Our study could increase the knowledge of the pathogenesis of hepatic fibrosis and might be applied to the diagnosis and treatment of patients.

Background
The activation of hepatic stellate cells is a key event in pathogenesis of liver fibrosis.

Research frontiers
In recent studies, various factors which were involved in the proliferation, apoptosis, ageing and extracellular matrix secretion were found to be contributive to the activation process of hepatic stellate cells.

Innovations and breakthroughs
This study found that embryonic liver fordin (ELF) was involved in hepatic stellate cells activation by regulating glucose metabolism, which was a novel aspect in the area of liver fibrosis.

Applications
The glucose metabolism of activated hepatic stellate cells provides us a new vision in the diagnosis and therapy of liver fibrosis.

Peer-review
In this study, the authors found that the ELF was involved in the couples of signaling pathways to activate the glucose glycolysis of hepatic stellate cells during the liver fibrosis. The PI3K/Akt signaling and glucose glycolysis-related proteins were evaluated in this study.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
PD-1 pathway-mediated regulation of islet-specific CD4+ T cell subsets in autoimmune diabetes

Type 1 diabetes (T1D) is a CD4+ T cell-driven autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells. Clinical evidence and studies in non-obese diabetic (NOD) mice suggest that insulin is a major autoantigen. With this in mind, we developed insulin B10-23:IAg7 tetramer reagents to track insulin-specific CD4+ T cells in mice and interrogated the role of Programmed death-1 (PD-1) for peripheral tolerance. PD-1 is a T cell inhibitory receptor necessary to maintain tolerance and prevent T1D in NOD mice. PD-1 pathway inhibitors are increasingly used in the clinic for treating malignancies, and while many patients benefit, some develop adverse autoimmune events, including T1D. We therefore sought to understand the role of PD-1 in maintaining islet-specific tolerance in diabetes-resistant strains. B6.g7 mice express the same MHC Class II allele as NOD mice, have predominantly naïve insulin-specific CD4+ T cells in the periphery, and remain diabetes-free even after PD-1 pathway blockade. Here, we examined the trafficking potential of insulin-specific CD4+ T cells in NOD and B6.g7 mice with or without anti-PD-L1 treatment, and found that PD-L1 blockade preferentially increased the number of CD44highCXCR3+ insulin-specific cells in NOD but not B6.g7 mice. Additionally, we investigated whether pancreatic islets in NOD and B6.g7 mice expressed CXCL10, a lymphocyte homing chemokine and ligand for CXCR3. Anti-PD-L1 treated and control NOD mice had detectable CXCL10 expression in the islets, while B6.g7 islets did not. These data suggest that islet tolerance may be in part attributed to the pancreatic environment and in the absence of pancreas inflammation, chemotactic cytokines may be missing. This, together with our previous data showing that PD-1 pathway blockade preferentially affects effector but not anergic self-specific T cells has implications for the use of checkpoint blockade in treating tumor patients. Our work suggests that determining tumor- and self-specific CD4+ T cell activation status (naïve, effector or anergic) prior to initiation of immunotherapy would likely help to stratify individuals who would benefit from this therapy versus those who might have adverse effects or incomplete tumor control.

Introduction
Type 1 diabetes (T1D) is caused by the immune-mediated destruction of insulin-producing pancreatic beta cells in the islets of Langerhans [1] . An estimated 3 million people currently suffer from T1D in the United States alone. In the last decade, incidence has risen by 23% among individuals younger than 20 years of age, and this alarming trend is expected to continue [2] . Daily insulin injections are the standard of care, but they are not a cure. Due to artificial blood glucose regulation, T1D patients remain at an increased risk of heart and kidney disease, blindness and peripheral neuropathy [3][4][5] . These complications have a significant impact on the quality of life and longevity [6] . Islet transplantation is an attractive therapeutic approach, but requires immunosuppression. Understanding how islet-reactive lymphocytes are activated, escape peripheral tolerance, and cause disease is necessary to design antigen-specific therapies to cure T1D.
Clinical evidence as well as studies using the non-obese diabetic (NOD) mouse model of spontaneous T1D demonstrate that CD4 + and CD8 + T cells are critical for beta cell destruction [7][8][9][10][11][12][13] . While a case study described T1D onset in a patient with X-linked agammaglobulinemia [14] , new-onset T1D patients benefited from B cell depletion therapy, suggesting that B cells are also required for disease [15] . In fact, B cell-mediated autoantibody production against islet antigens precedes T1D onset and is currently the only immunological biomarker of disease progression [3] . Specifically, insulin autoantibody onset can predict time to overt T1D in mice [16] and to a lesser degree in humans. All patients who develop T1D before age 5 have insulin autoantibodies [17] , suggesting that insulin is a critical autoantigen. In NOD mice, as many as 90% of insulin-specific CD4 + T cells target insulin B 10-23 residue [18] . This peptide is required for T1D, as a single mutation in a T cell receptor contact site abrogates disease [19] . With that in mind, we and others developed insulin B 10-23 :MHC Class II tetramer reagents [20][21][22][23] to track insulin-specific CD4 + T cells during disease development and at onset in NOD mice, as well as interrogate the fate of this population in diabetes resistant B6.I-A g7 (B6.g7) mice to understand tolerance mechanisms in play [24,25] .
Programmed death-1 (PD-1) is a T cell inhibitory receptor, and it is highly expressed on recently activated effector T cells as well as chronically stimulated (anergic) CD4 + and (exhausted) CD8 + T cells, thus limiting their antiviral and antitumor activity [26][27][28] . Blocking PD-1 signaling has the potential to reinvigorate anergic or exhausted cells. This spurred the development of PD-1 pathway inhibitors (checkpoint blockade) for the treatment of advanced malignancies [29,30] . While some patients benefit from this treatment, it is unclear why others do not, or why some patients develop adverse events and proceed to develop autoimmune-like symptoms or overt autoimmunity, including T1D [31] . PD-1 SNPs have been shown to increase the risk of T1D development in several populations [32][33][34][35] , suggesting that at least in a subset of patients, PD-1 plays a critical role in maintaining islet tolerance. Deficiency in, or blocking PD-1 from interacting with its ligand programmed death ligand-1 (PD-L1), accelerates T1D onset in NOD mice ( Figure 1); [36][37][38][39][40] ). However, the role PD-1/PD-L1 pathway plays in islet tolerance maintenance in non-autoimmune prone mouse strains is unclear. To address this gap in knowledge, we sought to determine whether the PD-1 signaling pathway regulated islet-specific CD4 + T cells in mice of varying autoimmune susceptibilities, as well as whether anergy maintenance required continuous PD-1 signaling [25] .
To illustrate previous work combined with results presented here, we have generated a model figure depicting the relative number of antigen specific cells, expansion, trafficking to the pancreas, and islet destruction ( Figure 1). Compared to NOD mice ( Figure 1A), B6.g7 mice had a detectable population of insulin-specific CD4 + T cells in the secondary lymphoid organs (SLO), but these cells remained predominantly naïve throughout the lifetime of the animal ( Figure 1D) and expressed low levels of PD-1 [25] . PD-1 pathway blockade led to an increased number of activated insulin-specific CD4 + T cells in the pancreatic lymph node of both NOD ( Figure 1B, C) and B6.g7 mice ( Figure 1E), but B6.g7 mice remained diseaseand infiltrate-free [25] ( Figure 1E). PD-1/PD-L1 deficiency on a diabetes-prone background (NOD) led to accelerated disease as previously reported, and increased numbers of insulinspecific but not foreign antigen-specific CD4 + T cells in the SLO and pancreas [25] .
Since the majority of islet-specific CD4 + T cells in NOD mice are anergic [24] , we also investigated whether PD-1 pathway blockade restores function to these cells. Anergic cells were defined as cells producing little to no IFNγ and expressing CD73 and folate receptor 4 (FR4) on their surface [24,41] . Effector cells were defined as FR4 − CD73 − cells capable of producing IFNγ following stimulation [24,41] . Following anti-PD-L1 treatment, effector cells produced significantly more IFNγ on a per cell basis. While PD-1 blockade led to a modest increase in the amount of IFNγ produced by anergic cells, IFNγ production was still blunted in comparison to effector cells [25] . This suggested that PD-1 pathway blockade preferentially impacted the effector cell subset, and did not uniformly break T cell anergy.
In the current study, we evaluated the insulin-specific CD4 + T cell trafficking potential from NOD and B6.g7 mice with and without PD-1 pathway blockade. Collectively, our findings suggest that PD-1 blockade was not sufficient to result in B6.g7 lymphocyte homing to the pancreas due to lower expression of CXCR3 on insulin-specific CD4 + T cells, and an absence of a CXCL10 chemotactic gradient.

Materials and Methods
Mice NOD mice were purchased from Taconic (Germantown, NY). B6.g7, NOD.PD-1 −/− (PD-1 KO), and NOD.PD-L1 −/− (PD-L1 KO) mice were generated as described [25,42] and housed in specific-pathogen free conditions at the University of Minnesota. All animal experiments were approved by the Institutional Animal Care and Use Committee.

Detection of antigen-specific T cells
Insulin-specific CD4 + T cells were detected using insB 10-23r3 :I-A g7 tetramer reagent and dual color staining with magnetic enrichment as described [24,25] . Briefly, single cell suspensions were incubated for one hour at room temperature with 10 nM of tetramers conjugated to phycoerythrin (PE) and allophycocyanin (APC) in medium containing Fc receptor block (2.4G2) and 0.05% sodium azide. Spleen and non-draining lymph node samples were subjected to magnetic enrichment following a 30 minute incubation with both anti-PE and anti-APC microbeads at 4°C (Miltenyi Biotec). Single cell suspensions from the pancreas were isolated using collagenase P digestion (Roche) and discontinuous Percoll gradients (44%/67%) [24,25] . Following tetramer staining, single cell suspensions from pancreas and pancreatic lymph node were subjected to surface staining for flow cytometric analysis.

Results and Discussion
Emerging results from genome wide association studies point to PD-1 as a critical risk factor in T1D pathogenesis [32][33][34][35] . Additionally, PD-1 pathway inhibitors used to treat advanced malignancies have led to T1D development in several patients, indicating that at least in a subset of individuals, PD-1 is required for restraining islet-reactive T cells [31] . Increasing use of checkpoint blockade therapies warrants a better understanding of how PD-1 regulates islet-reactive CD4 + T cells in contexts of varying autoimmune susceptibilities. To this end, we examined insulin-specific CD4 + T cells following checkpoint blockade in diabetes-prone and diabetes-resistant mice expressing the same MHC class II molecule.
Genetic loss of PD-1 or PD-L1 in NOD mice led to an increased number of insulin-specific but not foreign antigen-specific CD4 + T cells [25] . Not only was there an increase in the abundance of insulin-specific cells in both PD-1 KO and PD-L1 KO mice, these cells exhibited increased expression of the activation marker CD44 compared to age-matched NOD mice (Figure 2). The number of CD44 high insulin-specific CD4 + T cells was significantly higher in the pancreatic lymph nodes of prediabetic (907.9±310.4 cells) and diabetic PD-1 KO mice (347.2±137.0 cells) (p<0.001 and p<0.01, respectively) compared to prediabetic NOD mice (117.0±29.5 cells) (Figure 2A). Similarly, diabetic PD-L1 KO mice had a significantly greater number of CD44 high insulin-specific CD4 + T cells (395.9±122.2 cells) compared to age-matched NOD mice (p <0.01) (Figure 2A). The number of activated insulin-specific CD4 + T cells in the spleen and non-draining lymph nodes (spl+non-dLNs) was not different across these strains ( Figure 2B). Additionally, CD44 expression on foreign antigen-specific CD4 + T cells was comparable (data not shown).
PD-1 pathway blockade led to an increase in the number of activated insulin-specific CD4 + T cells in the pancreatic lymph nodes of NOD and B6.g7 mice [25] (Figure 1B, E), but B6.g7 mice remained insulitis-and diabetes-free [25] (Figure 1E). We sought to determine whether anti-PD-L1 treatment altered the trafficking potential of insulin-specific cells in NOD or B6.g7 mice. Pancreas homing is regulated by the CXCR3-CXCL10 axis [43,44] . Therefore, we assessed the expression levels of CXCR3 on insulin-specific CD4 + T cells in NOD and B6.g7 mice that were treated with anti-PD-L1 or control at days -3 and -1 prior to harvest. Anti-PD-L1 led to a significant increase in the number of CD44 high CXCR3 + cells in the pancreatic lymph nodes of NOD (10.16±2.25 vs. 111.5±23.7, p<0.01), but not B6.g7 mice (2.09±0.55 vs. 3.78±1.62) ( Figure 3A). Additionally, the number of insulin-specific CD4 + T cells with the potential to traffic was higher in NOD pancreatic lymph nodes compared to B6.g7 in control (p <0.01) and anti-PD-L1 treated mice (p<0.001). While anti-PD-L1 treatment did not lead to a significant increase in CD44 high CXCR3 + cells in NOD spleen and nondraining lymph nodes, the number of cells with trafficking potential was larger than in B6.g7 mice (p<0.01 between control and anti-PD-L1 treated mice). The fold increase in the geometric mean florescence intensity (gMFI) of CXCR3 was higher on CD44 high insulin-specific cells after anti-PD-L1 treatment in the pancreatic lymph node of NOD mice (p<0.01) ( Figure 3B). These results suggest that NOD islet-reactive cells were more susceptible to PD-1 pathway blockade allowing enhanced activation (CD44) and greater ability to traffic to tissues with inflammation including the pancreas (CXCR3).
The inflammatory cytokine interferon gamma (IFNγ) is known to be important for disease onset [45,46] . CXCL10, or interferon regulated protein of 10Kd, is a chemokine that is made in response to IFNγ and inflammation [43,47] . We hypothesized that B6.g7 islets lacked chemotactic gradients necessary to recruit the autoreactive cells. To test this, we examined islet CXCL10 expression by histology to correlate the level of islet infiltration with chemokine production. CXCL10 was absent from B6.g7 islets even after PD-L1-blockade, while CXCL10 was readily detectible in the islets of age-matched treated and untreated NOD mice ( Figure 3C). Collectively, these findings indicate that there are at least two levels of islet tolerance: T cell-intrinsic and pancreas-microenvironment specific. We hypothesize that the initial wave of islet inflammation depends on CCL2, CCL5, and CXCL9 secreted in response to inflammatory cytokines such as IL-1β and TNF-α [48] , and that CCR2 + or CCR5 + T cell-derived IFNγ drives CXCL10 expression in pancreatic beta cells to recruit CXCR3 + insulin-specific T cells. Further studies are needed to determine whether altering the pancreatic environment in B6.g7 mice would be sufficient to allow for T cell infiltration, islet destruction and diabetes development.
Our previous work suggests that the susceptibility to PD-1 blockade-mediated functional restoration depends on the differentiation state of CD4 + and CD8 + T cells [25] . Effector, but not anergic autoreactive CD4 + and CD8 + T cells were reinvigorated after PD-1 blockade, suggesting that blocking PD-1 signaling alone is not sufficient to rescue effector function for all anergic cells [25] . Therefore, blocking the PD-1/PD-L1 pathway alone might not be sufficient to reinvigorate all anergic tumor-specific cells, but it also might not be sufficient to restore full effector cell function of previously anergic, autoreactive T cells and/or allow T cell homing to target organ(s). Determining tumor-and self-specific CD4 + T cell activation status prior to initiation of checkpoint blockade therapy would likely help to stratify individuals who would benefit from this therapy versus those who might have adverse effects or incomplete tumor control.

T1D
type 1 diabetes  In NOD mice, insulin-specific CD4 + T cells encounter antigen in the pancreatic lymph node, expand and acquire the potential to traffic to the pancreas between 3 and 5 weeks of age. Pancreatic inflammation becomes more severe as mice age and peaks at disease onset, between 10 and 30 weeks (15 weeks as illustrated here). (B) Anti-PD-L1 treatment at 4 weeks of age significantly enhances T cell activation, thus leading to accelerated diabetes onset within 20 days in 90% of treated animals [38] . (C) PD-1 pathway blockade at a later time point when existing insulitis is extensive (at 15 weeks for example) [25] , leads to a very rapid disease onset in NOD mice within 1-3 days [38] . (D) In B6.g7 mice, insulin-specific CD4 + T cells do not expand as mice age and ~80% of the islets remain uninfiltrated [25] . (E) PD-1 pathway blockade at 15 weeks of age leads to an increased number of activated insulin-specific CD4 + T cells in the pancreatic lymph node, but insulin reactive T cells do not traffic to the pancreas and the extent of islet inflammation does not change [25] .

Figure 2. NOD PD-1/PD-L1 deficiency leads to increased activation of insulin-specific CD4 + T cells
The frequency of activated (CD44 high ) insulin specific T cells was determined from (A) pancreatic lymph nodes and (B) Spleen and non-draining lymph nodes of 5-6 week old PD-1 KO (n=8), PD-L1 KO (n=4-7), and age-matched littermate NOD controls (n=22) by flow cytometry prior to or at disease onset ("DB"=diabetes) using insB 10-23r3 :I-A g7 tetramer reagents. Shown are compiled data from four independent experiments. NOD and B6.g7 mice were treated at day −3 and −1 with anti-PD-L1 (MIH6 clone; 250 μg/ mouse) or isotype control (Rat IgG2a; 250 μg/mouse). (A) The number of CD44 high CXCR3 + insulin-specific CD4 + T cells (identified by double-staining with insB 10-23r3 : I-A g7 -tetramers as described in Methods) in the secondary lymphoid organs of control and anti-PD-L1-treated NOD and B6.g7 mice. (B) Fold increase in geometric mean fluorescent intensity (gMFI) of CXCR3 after PD-L1 blockade from insulin-specific CD4 + T cells. Shown are compiled data from four independent experiments with n=2-3 per treatment group. (C) Immunofluorescent staining of CXCL10 expression in pancreas tissue from NOD and B6.g7 mice from anti-PD-L1 or isotype treated animals. Scale bar (white) corresponds to 50 μm. Images are representative of three independent experiments with >100 islets/treatment group.


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Involvement of human chorionic gonadotropin in regulating vasculogenic mimicry and hypoxia-inducible factor-1α expression in ovarian cancer cells

Background Human chorionic gonadotropin (hCG) can play a crucial role in angiogenesis. In the present study, we focused on hCG to gain insight into its potential effects on vasculogenic mimicry (VM) in ovarian cancer cells. Methods Ovarian cancer OVCAR-3 cells were incubated with different concentrations of recombinant hCG in 3-dimensional cultures. VM was identified by morphological observations and vascular endothelial cell marker detection in OVCAR-3 cells. Expression of hCG, hypoxia-inducible factor-1α (HIF-1α), and the endothelial cell markers CD31, VEGF, and factor VIII were detected by reverse transcription polymerase chain reaction and western blotting. The effect of hCG on endothelial cell-marker expression in ovarian cancer cells was further explored using small interfering RNA (siRNA) and plasmid-based approaches. Results Incubation of OVCAR-3 cells with recombinant hCG induced vessel-like network formation, which was accompanied by significant elevation of vascular marker expression. Attenuation of hCG expression by siRNA in OVCAR-3 cells suppressed the expression of endothelial cell markers and HIF-1α by tumour cells. Overexpression of hCG in OVCAR-3 cells resulted in increased expression of endothelial cell markers and HIF-1α. Conclusions HCG was crucial for changing the phenotype of OVCAR-3 cells to endothelial-like cells. The effect of hCG induction on VM in ovarian cancer cells is potentially associated with HIF-1α.

Background
The concept of vasculogenic mimicry (VM) was introduced in 1999 and was described as the unique ability of highly aggressive melanoma cells to obtain endothelial-like characteristics and form de novo vascular-like networks. The aggressive tumor cells have the potential to express vascular marker in this novel microcirculation [1]. Tumour cells have direct access to the bloodstream through the tumour cell-lined vessels and tend to haemorrhagic channels, similar to VM. Recently, we reported that ovarian cancer cells can express endothelium-associated genes to form vasculogenic-like networks in 3D gels in a microenvironment containing added hCG [12,13]. HCG belongs to a family of glycoprotein hormones characterized by a heterodimeric structure with an α-subunit non-covalently bound to the β-subunit, the latter being hormone specific [14]. Although β-hCG is normally expressed at detectable levels during pregnancy, it is also ectopically synthesized in trophoblastic and non-trophoblastic carcinomas of the colon, prostate, bladder, breast, lung, and ovaries [15,16]. β-hCG has recently been proposed as a biomarker of poor prognosis in cancer [17][18][19]. It has been suggested that placental hCG and vascular endothelial growth factor (VEGF) interact during formation of the placental vasculature [20]. Ectopically produced hCG has recently been found to exhibit angiogenic growth factor properties that are central to cancer progression [16]. β-HCG expression in cervical cancer is associated with the extent of tumour vascularisation [21]. Serum hCG levels have recently been linked to neo-vascularisation of non-seminomatous testicular germ cell tumours [22]. However, little has been reported regarding the effects of hCG on VM.
We hypothesised that hCG may play a crucial role in the development of VM in ovarian cancer. In this study, we explored the possible effects of hCG on VM in the hCG receptor-positive ovarian cancer cell line OVCAR-3 in a 3D angiogenesis system. OVCAR-3 cells were incubated with different concentrations of hCG to evaluate the influence of hCG on VM formation. HCG receptornegative ovarian cancer SKOV3 cells were used as a control. We identified VM by morphological observations and detected vascular marker expression. A small interfering RNA (siRNA) against hCG mRNA and a phCMV1derived hCG expression vector were used to gain insight into the potential effects of hCG on transendothelial differentiation and HIF-1α expression in OVCAR-3 cells.

Vascular cell marker expression and morphological flexibility induced by hCG in OVCAR-3 cells
OVCAR-3 cells were incubated in 3D gels with increasing concentrations of hCG (50, 500, or 5000 mU/ml) for 7 days. The expression of vascular cell markers in OVCAR-3 cells was analysed by reverse transcriptionpolymerase chain reaction (RT-PCR) and western blotting. As shown in Fig. 1a, the expression levels of CD31, VEGF, factor VIII mRNA and HIF-1α increased significantly in response to hCG treatment, in a dose-dependent manner, as did their respective protein-expression levels (Fig. 1b). The highest dose of hCG (5000 mU/ml) showed the most significant effect. We also found that the relative expression of hCG in OVCAR-3 cells significantly increased in response to hCG treatment in a dose-dependent manner, compared with that observed in unstimulated cells (Fig. 1a-d). However, hCG treatment did not significantly increase expression of the vascular cell marker in SKOV-3 cells.
OVCAR-3 cells displayed considerable plasticity in cell shape when embedded in the 3D matrix under hCG treatment when observed by light and scanning-electron microscopy. Tubular network and channel formation with OVCAR-3 cells were observed in the 3D gel exposed to 5000 mU/ml hCG (Fig. 1e). The effects in the 3D gel on exposure to 50 or 500 mU/ml hCG with respect to morphological changes were not obvious, compared with the appearance of untreated cells. SKOV-3 cells failed to form tubular networks or channels in the 3D gel, even when exposed to 5000 mU/ml hCG.

Inhibition of vascular marker and HIF-1α expression in OVCAR-3 cells by β-hCG siRNA
The specificity of the effect of hCG was further assessed by down-regulating β-hCG expression with siRNA. β-hCG siRNA specifically suppressed hCG expression. HCG mRNA expression decreased by 71.87 % and hCG protein expression decreased by 85.39 %. Our data showed that expression of vascular cell markers in OVCAR-3 cells was inhibited effectively by β-hCG siRNA. For example, expression of CD31, VEGF, factor VIII mRNA decreased by 57.36, 77.05, and 86.2 %, respectively, in OVCAR-3 cells transfected with β-hCG siRNA, compared with the negative control group. β-hCG siRNA also reduced CD31, VEGF, Factor VIII protein expression by 82.68, 71.05, and 69.05 %, respectively. HIF-1α mRNA and protein expression was also decreased by 69.53 and 70.61 %, respectively ( Fig. 2; p < 0.01).

Expression of vascular markers and HIF-1α in OVCAR-3 cells with up-regulated β-hCG expression
To further investigate the effect of hCG on the expression of vascular markers in ovarian cancer cells, OVCAR-3 cells were transfected with the phCMV1 vector expressing β-hCG. HCG mRNA and protein expression in transfected OVCAR-3 cells was verified by RT-PCR and western blot analysis. Compared with parental and vector control cells, a higher level of hCG expression was detected in transfectants overexpressing hCG. HCG expression increased by 6.5-fold at the mRNA level and 2.7-fold at the protein level. The angiogenic efficacy of hCG was evaluated by analysing the expression of vascular markers in OVCAR-3 cells overexpressing hCG. OVCAR-3 cells transfected with the phCMV1-hCGβ vector showed a significant increase in the expression Su et al. Cancer Cell Int (2016) 16:50 Fig. 2 Inhibition of hCG expression using siRNA resulted in suppressed vascular marker and HIF-1α expression. a, b Expression of hCG in OVCAR-3 cells was inhibited by siRNA targeting hCG mRNA, but not by a negative control siRNA. Compared with untransfected OVCAR-3 cells and mocktransfected OVCAR-3 cells, the expression of CD31, VEGF, factor VIII, and HIF-1α decreased in OVCAR-3 cells transfected with hCG siRNA. a mRNA expression of the vascular cell marker, HIF-1α and hCG was analysed by RT-PCR. b Protein expression of the vascular cell marker, HIF-1α and hCG was analysed by western blotting. c, d Band densities were quantified by densitometric analysis. Protein and mRNA content measured in 3 independent replicates was quantified and the data are presented as the mean ± SD. The data shown are presented after normalization with GAPDH bands and analysed by 1-way ANOVA. *p < 0.01 (See figure on previous page.) Fig. 1 Expression of vascular cell markers and hCG in and morphological flexibility of hCG-treated OVCAR-3 cells. a, b Expression levels of vascular markers CD31, VEGF, factor VIII, hCG, and HIF-1α were determined in OVCAR-3 cells exposed to 50, 500, or 5000 mU/ml hCG for 7 days. HCG treatment stimulated the expression of vascular markers and HIF-1α in OVCAR-3 cells in a dose-dependent manner. a The mRNA levels were analysed by RT-PCR. b Protein levels were detected by western blotting. c, d Band densities were quantified by densitometric analysis. Protein and mRNA content was quantified for 3 independent replicates and the data are presented as the mean ± SD. The data shown are presented after normalization with GAPDH expression and were analysed using 1-way ANOVA. *p < 0.01, # p < 0.05. e Light and scanning-electron microscopy observations showed tubular network and channel formation by OVCAR-3 cells in the 3D matrix after exposure to 5000 mU/ml hCG. Representative morphological changes are shown levels of CD31, VEGF, and factor VIII, compared with untransfected and mock-transfected cells. Overexpression of hCG also resulted in a twofold enhancement of HIF-1α expression (Fig. 3).

Expression of the hCG receptor (hCG-R) in OVCAR-3 cells
We confirmed that the hCG receptor was expressed in OVCAR-3 cells by confocal microscopy. The green fluorescence was localized to the periphery of OVCAR-3 cells (Fig. 4a). Expression of the hCG receptor in OVCAR-3 cells exposed to 50, 500, or 5000 mU/ml hCG for 7 days were analysed by RT-PCR and western blotting. As shown in Fig. 4b-e, treatment of OVCAR-3 cells with different hCG concentrations did not significantly affect expression of the hCG receptor.

Discussion
VM is the ability of aggressive cancer cells to acquire an altered phenotype and form a tumour cell-lined vasculature. The tumour cells can express endothelium-associated markers during VM. In tumour vessel channels non-endothelial cells have been found to express typical endothelial markers as seen in uveal melanoma cells which express the endothelial cell markers CD31 and CD34 [23]. Up-regulated expression of vascular markers and HIF-1α in OVCAR-3 cells transfected with the phCMV1 vector expressing β-hCG (phCMV1-hCGβ). a, b HCG expression increased significantly in transfected OVCAR-3 cells, as determined by RT-PCR and western blot analysis. Compared with untransfected and mock-transfected cells, both mRNA (a) and protein expression (b) of CD31, VEGF, Factor VIII, and HIF-1α increased significantly. c, d Band densities were quantified by densitometric analysis. Protein and mRNA content measured in 3 independent replicates was quantified and the data are presented as the mean ± SD. The data shown were normalized to GAPDH bands and analysed by 1-way ANOVA. *p < 0.01 Human glioma stem/progenitor cells can transdifferentiate into vascular endothelial cells (VECs) and express VECs markers including CD31, CD34, and vWF significantly under hypoxia [24]. Von Willebrand factor (VWF), a glycoprotein mainly secreted from endothelial cells, is a carrier protein of coagulation factor VIII (FVIII). Factor VIII-associated antigen are vasculogenic mimicry markers [25]. VEGF is a major angiogenesis regulator of human endothelial cells. VEGF appears to contribute to VM formation in some cancer types including ovarian carcinoma [26]. In our study, VM was identified by morphological observations and detection of vascular endothelial cell markers CD31, VEGF, Factor VIII in ovarian cancer cells.
It has been reported that ovarian cancer SKOV3 cells could differentiate into endothelial-like cells and form channels on scaffolds in a microenvironment with low oxygen tension [2]. Our previous findings showed that a microenvironment with hCG localized to scaffolds strongly induced VM in hCG receptor-positive ovarian cancer OVCAR-3 cells, even under normoxic conditions [12].
HCG is a heterodimeric hormone that is primarily produced by the placenta, but is also produced by other normal and cancer tissues at low levels [27,28]. The human epithelial ovarian cancer cell line OVCAR-3 not only synthesizes hCG, but also expresses hCG receptor on the cell membrane. HCG serves a role in angiogenesis both in vivo and in vitro by increasing capillary formation and endothelial cell migration [29][30][31]. Berndt et al. [32] demonstrated a direct angiogenic effect of hCG between blastocysts and the maternal endometrium in several experimental models. HCG can also facilitate trophoblast differentiation [33] and positively influence angiogenesis by inducing VEGF and matrix metalloproteinase 9 expression [34]. The angiogenic function of tumourderived hCG in VM has not been reported.
The angiogenic activity of hCG was investigated here by detecting expression differences in the vascular marker and morphological alterations in OVCAR-3 cells in 3D gels. The addition of exogenous hCG induced expression of vascular markers in OVCAR-3 in a dose-dependent manner, with a dose of 5000 mU/ml hCG in 3D gels Fig. 4 Expression of the hCG receptor (hCG-R) in the ovarian cancer cell line OVACR-3. a Confocal image of OVCAR-3 cells following immunofluorescence staining with an hCG-R antibody. Green fluorescence was localized to the periphery of OVCAR-3 cells. Blue fluorescence (PI) was used to demonstrate the nucleus. b, d Different concentrations of hCG did not significantly affect hCG-R expression in OVCAR-3 cells. b Protein expression of hCG-R in OVCAR-3 cells treated with hCG (0, 50, 500, or 5000 mU/ml) for 7 days was detected by western blot analysis. d Expression of hCG-R mRNA in OVCAR-3 treated with hCG (50, 500, or 5000 mU/ml) was detected by RT-PCR. c, e Band densities were quantified by densitometric analysis. The protein and mRNA content measured in 3 independent replicates was quantified and the data are presented as the mean ± SD. The data shown were normalized to GAPDH bands and analysed using 1-way ANOVA. p > 0. 05 showing the strongest influence on vessel-like tube formation by OVCAR-3 cells. These results indicated that hCG potentially affects VM.
In an effort to better understand the involvement of hCG in mediating VM in ovarian cancer cells, siRNA was used to block hCG expression and study its effect on the expression of vascular markers in OVCAR-3 cells. Transfection of the antisense hCG gene resulted in a significant inhibition of vascular cell marker expression in OVCAR-3 cells. OVCAR-3 cells were also transfected with the phCMV1 vector, which drove hCG overexpression and significantly increased vascular cell marker expression. Our data indicated that hCG promotes the trans-differentiation of OVCAR-3 cells into endothelial-like cells.
However, in hCG receptor-negative SKOV3 cells, exogenous hCG failed to induce VM formation. These data suggested that the hormone may act specifically through the hCG receptor. The activity of hCG is initiated by binding of hCG to its transmembrane glycoprotein receptor, which is a member of the G protein-coupled receptor superfamily [35]. Adenylate cyclase on the internal membrane is then stimulated to convert adenosine triphosphate into cyclic adenosine monophosphate [36]. Immunofluorescence staining, RT-PCR, and western blot data detected stable expression of the hCG receptor in the OVCAR-3 cell line. Although OVCAR-3 cells are positive for the hCG receptor, treatment of OVCAR-3 with increasing doses of exogenous hCG had no significant effect on expression of the hCG receptor, suggesting other possible regulatory pathways involved in the effect of hCG [37], which should be a subject of future studies.
We also investigated the effect of hCG on HIF-1α expression. Expression of HIF-1α in OVCAR-3 cells was up-regulated following hCG treatment or transfection with the phCMV1 vector, which drove overexpression of hCG. Conversely, attenuating hCG expression in OVCAR-3 cells via siRNA suppressed HIF-1α expression. HIF-1α is a key transcription factor that mediates responses to oxygen deprivation [38]. Hypoxia, an important feature of the tumour microenvironment, is known to mediate tumour VM through HIF-1α [2,4]. Driesche et al. [39] demonstrated that HIF-1α expression was induced by hCG in luteinizing granulosa cells under both hypoxic and normoxic conditions. Our present data indicated that hCG is an important regulator of HIF-1α and its downstream target, the vascular marker VEGF [40,41]. We propose that hCG may exert its angiogenic effect through the HIF-1α-VEGF pathway.

Conclusions
These results may offer new insights into the possible regulatory role of hCG in VM formation in ovarian cancer. The hCG receptor in OVCAR-3 ovarian cancer cells may potentially serve as a novel target in cancer therapy. Further studies are required to evaluate the signal transduction pathways involved in the activity of hCG in VM of ovarian cancer.

HCG treatment in 3D cultures
The human epithelial ovarian cancer cell lines OVCAR-3 and SKOV3 were purchased from the American Type Culture Collection (Manassas, VA). One hundred and fifty microliters of a co-mixture of Matrigel (Becton-Dickinson, Bedford, MA) and McCoy-5A/RPMI1640 (Gibco, Invitrogen, Carlsbad, CA) was dropped onto glass coverslips in 24-well culture plates and allowed to incubate for 30 min at 37 °C in a humidified 5 % CO 2 incubator. The medium contained 15 % foetal calf serum and was changed every 48 h. Tumour cells (1 × 10 5 ) were seeded onto the gels. Tumour cells were then exposed to different concentrations of recombinant hCG (50, 500, or 5000 mU/ml) for 7 days. HCG was obtained from Sigma (St Louis, MO, USA).

Western blot analysis
Mouse monoclonal antibodies against VEGF, factor VIII, β-hCG, hCG receptor, and HIF-1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A rabbit monoclonal antibody against CD31 was obtained from Bioworld (Dublin, OH). Cellular proteins were isolated after rinsing cells with ice-cold phosphatebuffered saline (PBS; pH 7.4) and lysing them on ice with a protein-extraction reagent. The proteins were then separated on an 8 % sodium dodecyl sulphide-Tris polyacrylamide gel. Transfer to a polyvinylidene fluoride membrane was performed at 0.27 mA for 2 h. The membranes were blocked overnight with 1× Trisbuffered saline containing 0.1 % Tween 20 and 5 % skim milk, followed by incubation with primary antibody (1:100) for 1 h and a horseradish peroxidase-conjugated secondary antibody (1:1000) for an additional 1 h at room temperature. Immunocomplexes were visualized by electrochemiluminescence. Protein expression was semi-quantified using a Tiannen imager and analysis system (Shanghai, China). Protein contents in the three independent replicates were respectively quantified and presented as mean ± SD.

Immunofluorescence staining and confocal microscopy observations
After fixing slides with paraformaldehyde, they were rinsed twice in PBST for 5 min. The slides were then immersed in 3 % hydrogen peroxide for 20 min to quench endogenous peroxidase activity. The specimens were pre-blocked for 30 min in bovine albumin serum. Subsequently, the slides were incubated with a rabbit polyclonal antibody against the hCG receptor (Santa Cruz, CA, USA) at a 1:100 dilution for 1 h at room temperature. After washing 3 times in PBS, the slides were incubated with a fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin (Santa Cruz, CA, USA) at a 1:100 dilution for 1 h at room temperature. Negative controls were prepared by replacing the primary antibody with Trisbuffered saline. Samples known to be positive for the hCG receptor served as positive controls. A Leica DM IRE2 confocal laser scanning system (oil immersion objectives 63´) with a helium ion/green neon laser (543 nm) was used. Images were collected and processed using Leica confocal software 2.0 and Adobe Photoshop 6.0.

Small interference RNA (siRNA)
HCG siRNAs were synthesized and ligated into the PGPU6/GFP/Neo vector by Jima Biologic Technology Co. (Shanghai, China). The sequence of pSilencer/β-hCG was 5′-CCCGAGGTATAAAGCCAGGTACA-3′. OVCAR-3 cells were seeded in 6-well plates and grown to 70-90 % confluency in the absence of antibiotics. Transfections were performed with 0.8 μg of the silencing plasmid PGPU6/GFP/Neo-β-hCG and 2 μl Lipofectamine ™ 2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer's recommended protocol. Control cells were mock-transfected. At 24 h post-transfection, the transfection efficiency was assessed by fluorescence microscopy, revealed that 80 % of the transfectants were positive for green fluorescent protein expression. Stably transfected cells were selected in G418 (0.4 mg/ml; Merck, Darmstadt, Germany) for approximately 2 weeks. The efficiency of β-hCG silencing was analysed by RT-PCR and western blotting.

Construction of the phCMV1 vector expressing β-hCG (phCMV1-hCGβ)
The recombinant phCMV1-hCGβ plasmid was constructed based on the phCMV1 vector (Gene Therapy System), which encodes the cytomegalovirus mediatedearly promoter plus intron A, followed by the SV40 polyA expression cassette. The vector expressing hCG was generated by cloning the sequences encoding hCG into the phCMV1 vector, using unique restriction endonuclease sites. PCR amplification of hCG was performed using the sense primer 5′-CGGAATTCTCC AAGGAGCCGCTTCGG-3′ and the antisense primer 5′-CGGGATCCTTGTGGGAGGATCGG-3′. OVCAR-3 cells were transfected with 2 μg of DNA using 6 μg Lipofectamine ™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacture's guidelines. The resistant clones were selected in G418 (800 μg/ml) for 7 days and expanded in 300 μg/ml G418.

Statistical analysis
All experiments were performed at least 3 times. The results are presented as the mean ± standard deviation (SD). The data were analysed using SPSS 16.0 for Windows software (SPSS, Inc., Chicago, IL). One-way analysis of variance (ANOVA) was performed to identify statistical differences.


extfieldsofstudy: Medicine Biology

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Complete Genome Sequence of a Reference Stock of Simian Immunodeficiency Virus RNA (SIVmac251/32H/L28) Determined by Deep Sequencing

A reference preparation for simian immunodeficiency virus (SIV) RNA nucleic acid assays was characterized by complete genome deep sequencing. The entire coding sequence and flanking long terminal repeats, including minority species, were determined. This information will inform SIV research investigations and aid evaluation and development of amplification assays for SIV RNA quantification.

pathogenesis studies of HIV/AIDS employ strains of simian immunodeficiency virus (SIV) in experimental design. In cynomolgus macaques (Macaca fascicularis), a high-titer bulk SIV positive plasma stock recovered at the peak of virus replication in vivo was developed into an SIV RNA reference panel (C1ϪC5), based on the SIVmac251/32H/L28 isolate (1,2). These represent commutable materials for determination of SIV viral load (2). The genetic composition of the highest viral concentration member of this panel (C1) was determined by deep sequencing analysis. The genetic composition of this SIV RNA reference material has not been described; here, we report the previously undescribed complete genome sequence using deep sequencing approaches, which are analogous to those previously reported for HIV-1 and HIV-2 (3,4).
Bar-coded libraries of amplicons were made using Nextera XT library preparation kits (Illumina), and equimolar amounts of each sample were sequenced in 250-bp paired-end MiSeq sequencing reactions. After removal of primer and adaptor sequences, de novo assembly was performed on quality-trimmed and paired reads using Geneious version 7 software. Resultant contiguous sequences were aligned with the SIVmm32H reference sequence (D01065), and a consensus sequence was generated. All reads were mapped against the consensus to establish read depth and minority species.
The sequence of the SIV RNA reference material described here is 10,276 bp long with a GϩC content of 44.0%, representing the complete coding sequence with nine open reading frames (gag, pol, vif, vpr, tat, rev, vpx, env, and nef), as well as the U5 region of the 5=-long terminal repeat (5=-LTR) and U3 and R regions of the 3=-LTR of the SIV genome. BLAST analysis (5) of the consensus RNA template-derived sequence further confirmed the highest similarity with the SIVmm32H proviral clone sequence (4) (total score 18,384, 98% identity, and 100% coverage). The mean read depth was 74,051.7-fold (Ϯ87,072.4 standard deviation) with a minimum of 16,285-fold. There are 39 positions with a minority nucleotide differing from the reference sequence with a frequency Ͼ3% and a base call accuracy of 99.9% (ϾPhred 30). In addition, there is one insertion event and one deletion event.
This is the first report of the complete genome sequence of the SIV RNA reference panel (CFAR catalogue no. 100092) derived from a viral RNA template, which will augment the development and evaluation of SIV RNA amplification assays applied in SIV vaccine and pathogenesis studies.
Nucleotide sequence accession number. The complete genome sequence of the C1 SIVmac251/32H/L28 reference used in the reference panel reported here has been deposited in GenBank under the accession number KU892415.

FUNDING INFORMATION
This work, including the efforts of Neil Almond and Neil Berry, was funded by Medical Research Council (MRC) (G060007).


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Targeting immune response with therapeutic vaccines in premalignant lesions and cervical cancer: hope or reality from clinical studies

ABSTRACT Human papillomavirus (HPV) is widely known as a cause of cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). HPVs related to cancer express two main oncogenes, i.e. E6 and E7, considered as tumorigenic genes; their integration into the host genome results in the abnormal regulation of cell cycle control. Due to their peculiarities, these oncogenes represent an excellent target for cancer immunotherapy. In this work the authors highlight the potential use of therapeutic vaccines as safe and effective pharmacological tools in cervical disease, focusing on vaccines that have reached the clinical trial phase. Many therapeutic HPV vaccines have been tested in clinical trials with promising results. Adoptive T-cell therapy showed clinical activity in a phase II trial involving advanced CC patients. A phase II randomized trial showed clinical activity of a nucleic acid-based vaccine in HPV16 or HPV18 positive CIN. Several trials involving peptide-protein-based vaccines and live-vector based vaccines demonstrated that these approaches are effective in CIN as well as in advanced CC patients. HPV therapeutic vaccines must be regarded as a therapeutic option in cervical disease. The synergic combination of HPV therapeutic vaccines with radiotherapy, chemotherapy, immunomodulators or immune checkpoint inhibitors opens a new and interesting scenario in this disease.

Introduction
Human papillomavirus (HPV) causes one-third of infectionassociated cancers. Although prophylactic vaccines provide protection against HPV-16 and HPV-18, the genotypes causing about 70% of cervical cancer, vaccination programs are disappointing in several countries, and many women remain at risk of developing cervical cancer. Moreover, the frequency of HPV-associated cancers continues to increase at anatomical sites other than the cervix, such as vagina, vulva, oropharynx, and anus, thus helping feed the problem [1].
HPV is a double-stranded DNA virus that exhibits unidirectional transcription. To date, more than 200 HPV genotypes have been identified, even if only few cause cancer [2] Six early proteins (E1, E2, E4, E5, E6, and E7) and two late, capsid proteins (L1 and L2) are synthesized by HPV during replication through the differentiating strata of epithelia. HPV virus can infect the basal epithelial cells in the cervical transformation zone, causing their proliferation and leading to epithelial hyperplasia or papillomatosis. E5, E6, and E7 are considered oncoproteins and are responsible for continuous cell proliferation, DNA amplification, and impairment of DNA repair mechanisms, with consequent accumulation of mutations, rearrangements, and aneuploidies. The integration of viral DNA into the host cell genome in selected cases is a crucial step, resulting in deletion of E2 gene and adjacent regions (frequently E4, E5, and L2 genes). The E2 deletion, a transcriptional regulator of E6 and E7, leads to overexpression of E6/E7 that, in turn, induce degradation of p53 and pRB, respectively. In addition, E6/E7 alter expression of genes involved in S phase of the cell cycle, interact with p21 and p27 that inhibits cyclin-dependent kinases regulating the transition from G1 to S phase, and interfere with proapoptotic functions of some proteins such as c-myc, BAK, p53. As a consequence, this complicate and finely tuned process may lead to malignant transformation [3][4][5][6].
Consequences of HPV persistent infection are squamous intraepithelial lesions (cervical intraepithelial neoplasia, CIN) that are considered as precursors of cancer, graded into three progressive risk group according to the proportion of epithelium affected by abnormalities: CIN1, CIN2, CIN3 [7]. CIN1 can evolve in a small percentage of cases into CIN2-3, with a continuous proliferation and an increase in chromosomal instability reflected by increasingly mitotic figures, nuclear abnormality and clumps of pyknotic cells [8,9]. In CIN3, these cell alterations are distributed throughout the full thickness of epithelium rendering histological diagnosis quite easy.
Currently, it is estimated that 32-43% of CIN2/3 lesions undergo spontaneous regression [10,11]. However, we still do not have prognostic biomarkers that can distinguish the likelihood of regression at the individual patient level.
Standard treatment for patients with CIN2/3 is surgical excision (cold knife conization or loop electrosurgical excision). It is to note that a considerable morbidity is associated with CIN treatment including low birth weight, pre-term births, increased deaths, and more caesarian section [12]. Thus, other therapeutic options are needed. Among the non-surgical hypothetical therapeutic options, several immunotherapeutic strategies have been explored in preclinical and clinical trials, with some promising results [13,14].
Prophylactic HPV vaccines act by inducing neutralizing antibodies against the L1 capsid proteins of HPV-16 and HPV-18 [15,16], whereas therapeutic vaccines generate cell-mediated HLA restricted adaptive T-cell immunity for killing virus infected cells. Since appreciable levels of L1/L2 capsid antigens are not expressed by cervical cancer cells, therapeutic vaccines need targets other than L1/L2, and since HPV E6 and E7 are necessary for the initiation and maintenance of malignant transformation, these antigens represent attractive targets for therapeutic vaccines. Several therapeutic vaccines, employing vector-based peptide or protein-based, nucleic acid-based, viral/bacterial vector based, and cell-based therapeutic vaccines targeting the HPV-16 E6 and/or E7 antigens have been tested in order to increase viral specific immune responses [17,18]. Achieving a valid immunotherapy against HPV-related pathologies has to face several obstacles including major histocompatibility complex (MHC) class I downregulation, impaired antigen-processing, homing of T-cell killer, and immunosuppression due to Treg infiltration, and secretion of immunosuppressive cytokines [14]. Immunosuppression is less effective in pre-cancer lesions rendering more efficacious immunotherapeutic intervention. However, recent discovery of monoclonal antibodies against immune check-points has completely changed the immunotherapy of tumors by altering the immunosuppressive milieu of cancer [19]. Pre-clinical data from mouse models on large already established HPV tumors indicate the extremely effectiveness of immunotherapy in combination with immune check-point blockade [20,21].
In this review, we report preliminary results and ongoing studies on therapeutic vaccines in pre-invasive and invasive cervical cancer, focusing on vaccines that have reached the clinical trial phase. Table 1 summarizes the main characteristics of these trials, while details on relevant ongoing clinical trials are reported in Table 2. Figure 1 shows methodologies for production and delivery of HPV therapeutic vaccines as well as their immunological activity.

Dendritic cell-based vaccines
Dendritic cells (DCs) have strong ability to initiate and control T-cell response, being ideal candidates for immunotherapy strategies. They can be divided into DCs pulsed with peptides/ proteins, or transduced with DNA or viral vectors encoding target antigen. A pilot study was carried out in 15 advanced cervical  patients treated with autologous monocyte-derived DCs pulsed  with recombinant HPV-16 E7 or HPV-18 E7 oncoprotein, showing T-cell response and good tolerability, but no objective responses were observed [22]. Other studies used a DC vaccine pulsed with recombinant HPV-16/18 E7 antigens and IL-2, or keyhole limpet hemocyianin (an immunologic tracer molecule), with strong immunological response but no clinical effect [23,24]. Since preliminary reports with DC cancer vaccines consisting of ex vivo generated DCs loaded with tumor antigens did not show clinical responses in advanced cancer patients, the combination with cytokine to favor DC maturation and antigen presentation have been tested in various tumors [25][26][27][28]. Thirty-two patients with HPV-16/18 positive advanced cervical cancer were treated with HPV-16 E6 (arm A) or HPV E7 peptide (arm B) pulsed on peripheral blood mononuclear cells (PBMCs) cultured for 5-7 days in GM-CSF and IL-4 (pre-immature dendritic cells), to obtain immune response against the relevant peptide on both arms. ELISPOT and 51Cr release assays showed immune response against the relevant peptide in 63% of the patients in arm A and in 58% of the patients in arm B. No objective responses were observed, and treatment was well tolerated. The administration of pre-immature DCs pulsed with HPV-16 E6 or E7 was feasible and induced a specific immune response, thus deserving further investigations [29].
Gene-transduced DCs vaccines represent a promising strategy, since HLA restriction may be bypassed, and several preclinical studies have been carried out with DC vaccines transduced with adenoviral vector carrying a mutated E7 protein with encouraging results in animal models [30].
Cytokine-induced killer (CIK) cells, generated from PBMCs by culturing in the presence of various cytokines, combined with DCs through a co-culture of DC and CIK, have the advantage of fast proliferation, high cytotoxicity and a broad tumor killing spectrum [31]. In early cervical cancer, a phase II randomized trial was performed in 79 surgery-treated, high-risk cervical cancer patients. They received as adjuvant treatment cisplatin alone versus cisplatin combined with DC-CIK cells. The immune function was significantly improved in the experimental arm (lymphocyte ratio and expression of perforin, GraB and CD107a of PBMCs), and the cumulative recurrence rate in the experimental arm was significantly lower than that in the control arm [32].
Despite some promising results DC-based therapies have several limitations, such as labor-intensive and expensive procedures and, moreover their short half-life and absence of proliferation limit long-lasting immune response.

Tumor-cells-based vaccines
Tumor-cell-based vaccines comprise delivery of whole tumor cells in order to stimulate immune system to recognize tumorassociated antigens, and are often genetically modified in vitro with genes encoding cytokines (IL-2, IL-12, GM-CSF) to increase immune response [33][34][35]. At present, the use of tumor cells (harvested from the patients) as vaccination is not considered in CIN/early stages of the disease due to safety concerns. Adoptive T-cell therapy (ACT) ACT allows a more rigorous control on the magnitude of the targeted response than tumor vaccination strategies. T-cells of a desired specificity and phenotype can be identified and expanded in vitro to obtain following reinfusion a number of antigen-specific T-cells that is 10-fold greater than those reachable by using current therapeutic vaccines regimens alone [36]. Recently, this ACT approach was utilized in chemotherapy-refractory metastatic cervical cancer epithelial malignancy. Tumor-infiltrating T-cells were collected from patients, expanded, and selected for HPV reactivity (HPV-TILs). Single infusion of these TILs induced objective responses in three out of nine patients and the presence of TILs in peripheral blood 1 month after treatment was associated with clinical response [37]. Although this technique cannot be easily performed, it may represent a dramatic improvement of advanced cervical cancer treatment where even new biological therapies failed to ameliorate the prognosis of these patients.

Peptide-protein-based vaccines
Synthetic peptide vaccines can be divided into two groups: synthetic long peptides and specific epitope (short) peptides. Peptide-based therapeutic vaccines have the advantages of stability, safety and easy production [38], even if the immunogenity is low, and there is the need of identifying specific epitopes of HPV antigens for different HLA haplotypes [39]. Synthetic long peptides-based vaccines were largely tested in experimental models, and some of them tested in clinical trials [40]. The use of synthetic long peptides has several advantages over short peptides. Notably, it does not require patient selection based on MHC profile. Recently, the interest toward synthetic long peptides-based vaccines has been strengthened because of the broad range of antigenic epitopes through inclusion of immunogenic peptides or peptides which guide CD4 + T-helper or CD8+ cytotoxic responses. Results in animal models showed immunologic responses and HPV-16 tumors eradication in vaccinated animals [41,42].
An interesting study in high-grade HPV-16 related vulvar intraepithelial neoplasia (VIN), where the spontaneous regression is less than 1.5%, was recently published and the authors investigated the immunogenicity and the efficacy of a synthetic long-peptide vaccine in 20 women. They received multiple three to four doses of vaccine (a mixture of long peptide from HPV-16 oncoprotein E6 and E7 in incomplete Freund's adjuvant). Toxicity was low, and at 12 months of follow-up 9 patients over 19 showed complete response (47%), which was maintained at 24 months. Moreover, patients with complete response had a stronger immunologic response [43]. A phase I trial with synthetic long peptide vaccine in advanced cervical cancer showed low toxicity and high immunogenicity [44]. Consequently, the HPV-16-synthetic long peptide vaccine was tested in 20 advanced/recurrent cervical cancer patients in a phase II trial, and HPV-16-specific T-cell response was analyzed. In 13 evaluable patients, a HPV-16-specific T-cell response was recorded after two to four vaccinations, and patients failing to have a specific response also did not develop immunity against other antigens, suggesting a decreased T-cell immunocompetence. Unfortunately, although the vaccine was well tolerated, the induced specific immune response did not result in regressions of the tumor [45]. A phase I/II study with therapeutic HPV-16 long peptide vaccine (ISA101) at different doses with or without IFN-alpha in combination with carboplatin and paclitaxel in advanced cervical cancer (NCT02128126) is ongoing to determine safety and immunomodulating effects. A phase I dose-escalation trial of a peptide-based HPV therapeutic vaccine with Candida skin test reagent as adjuvant (PepCan) was carried out in 24 CIN2/3 patients, showing very low toxicity, a satisfactory regression rate, and a significant immune response [46]. The phase II is ongoing (NCT02481414). HPV-16 E6/E7-derived epitopes are being investigated as liposome-encapsulated in CIN (NCT02065973). A phase I study is testing a HPV vaccine consisting of four HPV-16 peptides in combination with Candin (purified extract from Candida Albicans) to determine optimal dose, immunogenicity, maximum tolerated dose in women with HSIL lesions (NCT01653249).
A phase II study of multiple peptides cocktail vaccine for advanced cervical and ovarian patients was recently presented at American Society of Clinical Oncology Meeting, in May 2015 [47]. In 21 cervical cancer patients they observed 2 complete responses, a median overall survival of 15.4 months and a good tolerability. A phase I trial is evaluating safety and efficacy of cyclophosphamide combined with specific epitope cocktail and IL-2 for advanced cancers including cervical cancers (NCT00676949).
The use of epitope-specific short peptides-based vaccines aims at ensuring precisely targeted cytotoxic T-lymphocyte (CTL) responses through immunization with specific epitopes, and it is well known how linear short peptide epitopes can induce cytotoxic responses. This kind of vaccine is restricted to patients of a given HLA type; thus, preliminary HLA typing is needed before vaccination. Some epitope-specific therapeutic HPV vaccines have been tested in clinical trials, with low clinical responses in VIN or CIN [48,49], even employing new delivery system [50], while other phase I trials are ongoing also in other diseases (NCT00257738; NCT01653249).
The advantage of protein-based vaccines over peptidebased is that the former can circumvent the MHC specificity limitation. However, a potential disadvantage of proteins vaccine is that they may induce antibody responses rather than CTL responses. To avoid this disadvantage and to improve immunogenicity, adjuvant have been added into vaccines and/or proteins for therapeutic vaccination have been linked to molecules able to better induce APC presentation.
Various protein vaccines have moved to clinical trials. Few protein-based vaccines have reached the clinical phase [17,51]. Immunization with HPV-16 E6/E7 fusion protein mixed with the ISOMATRIX adjuvant was shown to be safe and immunogenic in a Phase I study. In addition, enhanced CD8+ T-cell responses to both E6 and E7 were recorded in vaccinated patients versus placebo recipients [52]. A protein vaccine, TA-CIN, consisting of a fusion of HPV-16 L2, E6, and E7, was proved to be safe and able to elicit humoral response in all the treated women and cellular (T-cell specific) immunity in a subset of them [53]. A mutated HPV-16 E7 fused to part of Haemophilus influenzae protein D, mixed with adjuvant AS02B (GSK proprietary formulation) has been evaluated in and was shown to induce significant E7-specific CTL responses in Phase I/II clinical trials on CIN1-CIN3 [54]. A complete regression was reported in 22% of CIN3 patients in a phase II trial [55] employing HspE7 (a fusion protein consisting of HPV-16 E7 and Hsp65 from Mycobacterium Bovis -SNG00101). Another trial using the same vaccine in 20 CIN3 patients reported 35% of complete regression and correlation with immune response [56]. A phase II randomized (vs. standard surgical treatment) study with SGN00101 (HSP-E7) fusion protein is being tested in women with CIN3 (NCT00054041).

DNA-based vaccines
DNA-based vaccines are safe, can be easily produced, and are capable of inducing both CTL and Th immunity, and also B-cell immunity; moreover, they can be administered repeatedly for long-term protection, even if they lack in inducing potent antigen specific cellular response because of immune tolerance against self-antigens [57]. To increase immunogenicity, encoding xenogenic version of antigens, fusions of antigens with T-cells activating molecules, priming with DNA vectors followed by boosting with viral vectors, or the use of immunomodulators have been investigated.
A DNA plasmid (ZYC101) was tested in a phase I trial in 12 patients with anal inthraepithelial neoplasia, with a broad antigen-specific immune response [58]. The same vaccine was tested in 15 CIN2/3 patients, with five patients showing complete histological regression [59]. More interestingly, the new form of the vaccine (ZYC101a, including HPV-16/18 E6and E7-derived CTL epitopes), was employed in a randomized, double blind, phase II trial, enrolling 127 CIN2/3 patients, with a trend in the experimental arm for complete regression, and a statistically significant difference in very young patients [60]. A phase I-II ongoing study is testing safety and immunogenicity of a DNA vaccine, VB10.16 in women with CIN3 (NCT02529930). Another DNA vaccine targeting E7, pNGVLa-Sig/E7(detox)-Hsp70, was employed in a phase I-II trial in CIN2/3 patients. The E7 sequence was fused to a chaperone (Hsp70 from Mycobacterium tuberculosis) to enhance APC uptake, processing and presentation. Significant immune response was observed in eight patients, and a complete regression was observed in three patients [61]. More recently a phase I trial with sequential pNGVLa-Sig/E7(detox)-Hsp70 with a recombinant vaccine virus encoding E6/E7 fusion protein, with or without imiquimod, an immunomodulator, was tested in 12 patients with CIN3, and five of them showed complete regression [62]. The study is ongoing (NCT00788164). Another ongoing study is testing different routes of administration of pnGVL4a-CRT/E7 (Detox) in patients with CIN2/3 (NCT00988559).
Recently, a mixture of two plasmids encoding HPV-16/18 E6/ E7 antigens, VGX-3100 vaccine, administered by electroporation, is being tested in phase I-II trials. A phase IIb randomized, double-blind placebo controlled trial (NCT01304524) tested VGX-3100 in CIN2/3 patients. Among 107 evaluable patients, 53 of the treated patients (49.5%) and 11 (30.6%) of the placebo arm showed complete regression (p = 0.034). Toxicity was low, with mild injection-site reactions in almost all the patients, and erythema significantly more common in the vaccine arm, so the VGX-3100, inducing both T-cell and antibody response to E6 and E7, is the first therapeutic vaccine to show efficacy against HPV-16 and HPV-18 related CIN2/3 [63]. This study represents a proof of principle for a therapeutic approaches distinct from surgery. Another study is ongoing, testing VGX-3100, administered i.m. by electroporation with IL-2-12-coding construct (INO-9012) and optionally combined with radiotherapy (NCT02172911). A naked plasmid coding for HPV-16 and 18 E6/ E7 (GX-188E) and for fms-like tyrosine kinase-3 ligand (FLT3LG) administered i.m. by electroporation is being tested for safety in a phase I study in patients with CIN3 (NCT01634503), and with different doses in a phase I-II randomized study in patients with HPV-16 or 18 related CIN3 (NCT02139267). Another ongoing study with the same vaccine is a prospective, multicenter, observational, follow-up clinical study to determine recurrence of CIN and long-term safety (NCT02411019).

RNA-based vaccines
RNA-based vaccines are created using naked RNA replicons derived from alpha-viruses to promote antigen-specific immune response. The replicon-based vectors can replicate in a wide range of cell types, with different expression of antigens. These RNA replicons are less stable than DNA. A combined approach with DNA-launched RNA replicon, termed 'suicidal' DNA was developed for HPV vaccine in preclinical models [64]. The newest mRNA-based vaccines have also been developed and the RNActive ® vaccine platform from CureVac (Tübingen, Germany) is in clinical trials for prostate and non-small cell lung cancer. RNActive vaccines were well tolerated and induced long lasting, humoral and cellular immune responses. However, information about clinical testing on HPV-associate cancers is lacking.

Viral vector vaccines
Viral vector-based vaccines have high infection efficiency and high antigens expression, encoded by the virus in the infected cells. Many viral vectors, such as adenoviruses, alphaviruses, vaccinia viruses, fowlpox viruses have been tested, and the most interesting are vaccinia virus-based vaccines [65,66]. The first clinical trial of a live recombinant vaccinia virus expressing HPV-16 and -18 E6/E7 proteins was a phase I-II trial where eight advanced cervical cancer patients received a single dose of vaccine. No significant side-effects were observed, and 3 out of 8 patients had an HPV-specific antibody response [66]. A study tested a vaccinia vector-based vaccine expressing modified forms of HPV-16 and -18 E6 and E7 proteins (TA-HPV) in 29 patients with stage IB-IIA prior surgery, and specific CTLs and serologic response were found in a subset of patients [65]. Further positive results were observed in VIN and in VAIN.
The MVA-E2 viral-based vaccine is a modified Vaccinia Ankara virus, an attenuated replicon-deficient vaccinia strain, expressing E2, and a phase I-II study in 34 CIN2/3 patients showed complete regression in 20 patients, with the development of HPV-specific antibodies [67]. In another study 21 patients with CIN2/3 were treated with TG4001, a modified vaccinia Ankara viral vector encoding HPV-16 E6/E7 and IL-2, and 10 (48%) patients had clinical response at month 6, with seven complete responses, warranting further development of clinical trials [68]. A randomized phase II study (NCT01022346) is testing TG4001, and preliminary results reported significant differences between vaccine and placebo in a press release [69]. Twelve patients with HPV-16 associated CIN2/3 have been treated with peripheral vaccination with a heterologous DNA primerecombinant vaccinia vector-based boost vaccination regimen before surgical resection in a phase I study. The schema included two priming vaccination with a DNA vaccine expressing HPV-16 E7, following by a recombinant vaccinia boost expressing HPV-16 and -18 E6/E7. Three dose levels have been evaluated (cohort 1, 2, 3). Toxicity was low, mostly consisting in injection site reactions, and one out of three patients in each of the one and two cohorts had complete regression of the lesion at surgery, whereas at the highest dose level, three out of six patients had complete regression [62].
Adenoviral vector (rAd)-based vaccines showed immunological response in preclinical studies, but to date no therapeutic effects have been observed [70]. Recombinant adenovirus-p53 combined with chemotherapy was tested in a phase II study presented at American Society of Clinical Oncology May 2013 [71]. Forty patients received chemotherapy plus rAd-p53 or chemotherapy alone; the response rate was 95% versus 75%, and the overall 1-year survival rates were 90% and 65%, respectively, without serious adverse events related to rAD-p53.

Bacterial vector vaccines
Several bacterial vectors have been tested as therapeutic vaccines, such as Listeria monocytogenes, Lactobacillus plantarum, Lactococcus lactis, with the Listeria-based being the most extensively studied [72]. A phase I trial tested the tolerability of the Listeria-based vaccine (Lm-LLO-E7) in 15 patients with advanced cervical cancer, with some severe (grade 3) reactions in 40% of the patients, and 1 partial response [73].
The results of a prospective randomized trial employing a Listeria Monocytogenes-based vector vaccine (ADXS11-001) in 110 advanced cervical cancer patients were presented at the ASCO meeting in June 2014. Patients were randomized to three doses of vaccines or four doses with weekly cisplatin. Final 12-month's overall survival was 36%, 18-month's overall survival was 28%, response rate was 11% with six complete responses, and the authors reported 2% of grade 3 adverse events. No benefit was observed from the addition of cisplatin [74]. The NRG0265 phase II study with multiple doses of ADXS11-001 is currently ongoing [75, NCT01266460], without cisplatin, and preliminary results showed in 26 evaluable patients receiving at least one dose a 12-month overall survival of 38.5% [76]. Other trials with ADXS11-001 are ongoing in CIN2/3 or cervical cancer, and an international phase III study (AIM2CERV) of ADXS11-001 as adjuvant treatment of high-risk, locally advanced cervical cancer is under development in collaboration with Gynecologic Oncology Group.

Conclusions
Immunotherapy is one of the most promising strategies for cancer treatment. In cervical cancer and its precursors, the use of therapeutic vaccines was associated with the regression of premalignant lesions and some clinical benefit in cancer patients. To mitigate the immunosuppressive effects of cancer microenvironment, combinatorial approaches, such as radiotherapy, chemotherapy, immunomodulators, immune checkpoint inhibitors, are being tested in cervical cancer patients. Indeed, although chemotherapy and immunotherapy have not shown to be curative as single treatment modalities in the advanced setting, growing evidence suggests a synergistic activity with immunological approaches [77].
The available literature data suggest that therapeutic vaccines for pre-neoplasia and cancer of cervix are reality (i.e. synthetic long peptide or electroporated DNA vaccines). The improvement of all therapeutic strategies and the identification of their optimal combination opens a new scenario in the treatment of cervical cancer and pre-neoplastic lesions.

Expert commentary
About 20% of human cancers are linked to infectious diseases and among them one-third of tumors are caused by HPV. The observation that HPVs related to cancer express two main oncogenes, i.e. E6 and E7, that are essential for transformation and maintenance of transformed status, makes these oncogenes ideal targets of cancer immunotherapy. E6 and E7 can be considered the best tumor-associated antigen, and many different therapeutic vaccines have been produced and employed in clinical trials for cervical cancer and preneoplastic lesions. Some of these trials have demonstrated not only safety and immunological responses to therapeutic HPV vaccines but also their clinical efficacy. Thus, HPV therapeutic vaccines must be regarded as a therapeutic option together with other currently available therapies.

Five-year view
The HPV-E6 and -E7 antigens represent the ideal targets for therapeutic vaccines in premalignant and malignant cervical lesions. Looking to the future, applying new technologies (i.e. omics) to better define HPV infections that evolve to CIN and cervical cancer could enhance the efficacy of the vaccines by improving target selection and by reducing unwanted side effects. Since at present effective treatments are lacking mainly in advanced cervical disease, the next 5 years will be focused also on how to combine therapeutic vaccination with other conventional treatment strategies, such as chemotherapy, radiation, biological agents, and other immunologic approaches. In particular, check-point inhibitors, empowering any ongoing anti-cancer immune response that might have been too weak or exhausted, have shown extremely encouraging clinical activity. Monoclonal antibodies (mAb) interfering with CTLA-4-CD80/86, PD-1/PD-L1, TIM-3-GAL9, and LAG3-MHC-II belong to this category of check-point inhibitors and some of them are already US FDA approved for melanoma cancer treatment [20]. As the role of immunotherapy for the treatment of premalignant lesions and cervical cancer continues to evolve, several clinical trials are ongoing and in development, and further studies on the immune cellular and molecular mechanisms of action and on preclinical models are needed to better elucidate immunological background and to explore the optimal integration among treatments and combination immunotherapies. For the next 5 years, the combination or sequential application of immunotherapic agents will be the front line in the battle against cancer, including cervical cancer and its precursor. However, toxicities and prohibitive costs of immunostimulatory mAb are serious limits to combination immunotherapy and researches must be also focused on overcoming these obstacles.

Key issues
• HPVs linked to cancer express two main oncogenes, E6 and E7, that represent an excellent target for cancer immunotherapy. • Many therapeutic vaccines have been tested in order to increase viral specific immune responses, showing an acceptable safety profile and clinical efficacy. • In a phase II trial, patients with chemotherapy-refractory metastatic cervical cancer epithelial malignancy were treated with adoptive T-cell therapy. Despite this technique is technically difficult, it showed some clinical activity.
• In patients with HPV16 or HPV18 positive CIN, a phase II randomized trial showed clinical activity of a nucleic acidbased vaccine. • Multiple trials involving peptide-protein-based vaccines and live-vector based vaccines demonstrated that this approaches may be effective in CIN as well as in advanced cervical cancer patients. • HPV therapeutic vaccines is one of the most promising strategies for cancer treatment, showing regression of premalignant cervical lesions and some clinical activity in cervical cancer patients.

Financial & competing interests disclosure
This work was funded in part by AIRC IG 12916 and by Project of Mutual Collaboration of the Planning Department of the National Health Service of Italian Ministry of Health and Regina Elena National Cancer Institute for Implementation of HPV-UNIT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.