Structural Framework for Analysis of CD4+ T-Cell Epitope Dominance in Viral Fusion Proteins.

Antigen conformation shapes CD4+ T-cell specificity through mechanisms of antigen processing, and the consequences for immunity may rival those from conformational effects on antibody specificity. CD4+ T cells initiate and control immunity to pathogens and cancer and are at least partly responsible for immunopathology associated with infection, autoimmunity, and allergy. The primary trigger for CD4+ T-cell maturation is the presentation of an epitope peptide in the MHC class II antigen-presenting protein (MHCII), most commonly on an activated dendritic cell, and then the T-cell responses are recalled by subsequent presentations of the epitope peptide by the same or other antigen-presenting cells. Peptide presentation depends on the proteolytic fragmentation of the antigen in an endosomal/lysosomal compartment and concomitant loading of the fragments into the MHCII, a multistep mechanism called antigen processing and presentation. Although the role of peptide affinity for MHCII has been well studied, the role of proteolytic fragmentation has received less attention. In this Perspective, we will briefly summarize evidence that antigen resistance to unfolding and proteolytic fragmentation shapes the specificity of the CD4+ T-cell response to selected viral envelope proteins, identify several remarkable examples in which the immunodominant CD4+ epitopes most likely depend on the interaction of processing machinery with antigen conformation, and outline how knowledge of antigen conformation can inform future efforts to design vaccines.

Metabolic Reprogramming of Glioblastoma Cells during HCMV Infection Induces Secretome-Mediated Paracrine Effects in the Microenvironment.

Glioblastoma (GBM) is an aggressive primary central nervous system neoplasia with limited therapeutic options and poor prognosis. Following reports of cytomegalovirus (HCMV) in GBM tumors, the anti-viral drug Valganciclovir was administered and found to significantly increase the longevity of GBM patients. While these findings suggest a role for HCMV in GBM, the relationship between them is not clear and remains controversial. Treatment with anti-viral drugs may prove clinically useful; however, their results do not explain the underlying mechanism between HCMV infection and GBM progression. We hypothesized that HCMV infection would metabolically reprogram GBM cells and that these changes would allow for increased tumor progression. We infected LN-18 GBM cells and employed a Seahorse Bioanalyzer to characterize cellular metabolism. Increased mitochondrial respiration and glycolytic rates were observed following infection. These changes were accompanied by elevated production of reactive oxygen species and lactate. Due to lactate's numerous tumor-promoting effects, we examined the impact of paracrine signaling of HCMV-infected GBM cells on uninfected stromal cells. Our results indicated that, independent of viral transmission, the secretome of HCMV-infected GBM cells was able to alter the expression of key metabolic proteins and epigenetic markers. This suggests a mechanism of action where reprogramming of GBM cells alters the surrounding tumor microenvironment to be permissive to tumor progression in a manner akin to the Reverse-Warburg Effect. Overall, this suggests a potential oncomodulatory role for HCMV in the context of GBM.

Unique Transcriptome Changes in Peripheral B Cells Revealed by Comparing Age Groups From Naive or Vaccinated Mice, Including snoRNA and Cdkn2a.

Aging is associated with a decline in immune function that is not fully understood including vaccine failure. Here we report transcriptomic analysis on B cells from naive or influenza-vaccinated mice of 3 ages: young (15-23 weeks), middle-aged (63-81 weeks), and old mice (103-119 weeks). Our goal was expression profiling of B cells by age and history of vaccination to identify novel changes at the transcriptome level. We observed waning vaccine responses with age. In B cell transcripts, age and vaccination history were both important with notable differences observed in conducted analyses (eg, principal component, gene set enrichment, differentially expressed [DE] genes, and canonical pathways). Only 39 genes were significantly DE with age irrespective of vaccine history. This included age-related changes to box C/D small nucleolar (sno) RNAs, Snord123 and Snord1a. Box C/D snoRNAs regulate rRNAs through methylation and are linked to neurodegenerative, inflammatory, and cancer diseases but not specifically B cells or age. Canonical pathway changes implicated with age irrespective of vaccination history included EIF2, mTOR signaling, p53, Paxillin, and Tec kinase signaling pathways as well as cell cycle checkpoint. Importantly, we identified DE genes and pathways that were progressively altered starting in middle-age (eg, signaling by Rho family GTPases) or only altered in middle-age (eg, sphingosine-1-phosphate signaling), despite minimal differences in the ability of these mice to respond to vaccination compared to younger mice. Our results indicate the importance of vaccination or immune stimulation and analyses of multiple age ranges for aging B cell studies and validate an experimental model for future studies.

The p53/Adipose-Tissue/Cancer Nexus.

Obesity and the resultant metabolic complications have been associated with an increased risk of cancer. In addition to the systemic metabolic disturbances in obesity that are associated with cancer initiation and progression, the presence of adipose tissue in the tumor microenvironment (TME) contributes significantly to malignancy through direct cell-cell interaction or paracrine signaling. This chronic inflammatory state can be maintained by p53-associated mechanisms. Increased p53 levels that are observed in obesity exacerbate the release of inflammatory cytokines that fuel cancer initiation and progression. Dysregulated adipose tissue signaling from the TME can reprogram tumor cell metabolism. The links between p53, cellular metabolism and adipose tissue dysfunction and how they relate to cancer, will be presented in this review.

Regulation of Wnt/ß-catenin signaling by herpesviruses.

The Wnt/ß-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/ß-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/ß-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/ß-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/ß-catenin pathway to promote a virus-centric agenda.

Human cytomegalovirus infection dysregulates the canonical Wnt/ß-catenin signaling pathway.

Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected in utero. During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/ß-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/ß-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of ß-catenin and expression of ß-catenin target genes in these cells. HCMV infection leads to ß-catenin protein accumulation in a discrete juxtanuclear region. Levels of ß-catenin in membrane-associated and cytosolic pools, as well as nuclear ß-catenin, are reduced after infection; while transcription of the ß-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/ß-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.

Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1.

The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells.

Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

Toll-like receptors on human mesenchymal stem cells drive their migration and immunomodulating responses.

Adult human bone marrow-derived mesenchymal stem cells (hMSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. To achieve the desired clinical outcomes for this strategy requires a better understanding of the mechanisms that drive the recruitment, migration, and engraftment of hMSCs to the targeted tissues. It is known that hMSCs are recruited to sites of stress or inflammation to fulfill their repair function. It is recognized that toll-like receptors (TLRs) mediate stress responses of other bone marrow-derived cells. This study explored the role of TLRs in mediating stress responses of hMSCs. Accordingly, the presence of TLRs in hMSCs was initially established by reverse transcription-polymerase chain reaction assays. Flow cytometry and fluorescence immunocytochemical analyses confirmed these findings. The stimulation of hMSCs with TLR agonists led to the activation of downstream signaling pathways, including nuclear factor kappaB, AKT, and MAPK. Consequently, activation of these pathways triggered the induction and secretion of cytokines, chemokines, and related TLR gene products as established from cDNA array, immunoassay, and cytokine antibody array analyses. Interestingly, the unique patterns of affected genes, cytokines, and chemokines measured identify these receptors as critical players in the clinically established immunomodulation observed for hMSCs. Lastly, hMSC migration was promoted by TLR ligand exposure as demonstrated by transwell migration assays. Conversely, disruption of TLRs by neutralizing TLR antibodies compromised hMSC migration. This study defines a novel TLR-driven stress and immune modulating response for hMSCs that is critical to consider in the design of stem cell-based therapies.

Ovarian cancers overexpress the antimicrobial protein hCAP-18 and its derivative LL-37 increases ovarian cancer cell proliferation and invasion.

The role of the pro-inflammatory peptide, LL-37, and its pro-form, human cationic antimicrobial protein 18 (hCAP-18), in cancer development and progression is poorly understood. In damaged and inflamed tissue, LL-37 functions as a chemoattractant, mitogen and pro-angiogenic factor suggesting that the peptide may potentiate tumor progression. The aim of this study was to characterize the distribution of hCAP-18/LL-37 in normal and cancerous ovarian tissue and to examine the effects of LL-37 on ovarian cancer cells. Expression of hCAP-18/LL-37 was localized to immune and granulosa cells of normal ovarian tissue. By contrast, ovarian tumors displayed significantly higher levels of hCAP-18/LL-37 where expression was observed in tumor and stromal cells. Protein expression was statistically compared to the degree of immune cell infiltration and microvessel density in epithelial-derived ovarian tumors and a significant correlation was observed for both. It was demonstrated that ovarian tumor tissue lysates and ovarian cancer cell lines express hCAP-18/LL-37. Treatment of ovarian cancer cell lines with recombinant LL-37 stimulated proliferation, chemotaxis, invasion and matrix metalloproteinase expression. These data demonstrate for the first time that hCAP-18/LL-37 is significantly overexpressed in ovarian tumors and suggest LL-37 may contribute to ovarian tumorigenesis through direct stimulation of tumor cells, initiation of angiogenesis and recruitment of immune cells. These data provide further evidence of the existing relationship between pro-inflammatory molecules and ovarian cancer progression.

Erythropoietin, a hypoxia-regulated factor, elicits a pro-angiogenic program in human mesenchymal stem cells.

OBJECTIVE: The ability of erythropoietin (EPO) to elicit a pro-angiogenic effect on human mesenchymal stem cells (hMSC) was tested. hMSC are currently under study as therapeutic delivery agents that target tumor vessels. Hypoxia favors the differentiation of hMSC towards a pro-angiogenic program. However, the classical angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor, are not fully capable of restoring this effect. The hypoxia-regulated factor, EPO, induces angiogenesis in endothelial cells. Here, EPO's pro-angiogenic effect on hMSC was analyzed. METHODS: hMSC were tested for EPO receptor expression by western blot, immunofluorescence, and flow cytometry assays. Downstream receptor signaling components JAK and STAT were measured by standard assays. Pro-angiogenesis effects mediated by EPO treatment of hMSC were measured by proliferation, cytokine, or pro-angiogenesis factor secretion, metalloprotease activation, migration, invasion, wound healing, and tubule formation assays. RESULTS: hMSC express the cognate EPO receptor and are capable of promoting angiogenesis following EPO treatment in all the angiogenesis assays tested. EPO-treated hMSC proliferate and secrete pro-angiogenesis factors more readily than untreated hMSC. EPO leads to increased hMSC chemotaxis, migration, and activation of matrix metalloprotease-2. This treatment causes greater recruitment of vessels as measured in an in vivo angiogenesis assay. CONCLUSION: EPO is capable of eliciting a pro-angiogenesis program in hMSC that instigates secretion of angiogenic factors and the subsequent recruitment of endothelium. This study defines a novel mechanism for tumor cell recruitment of blood vessels that is important to consider in the design of stem cell-based therapies.

Integrin-linked kinase: a hypoxia-induced anti-apoptotic factor exploited by cancer cells.

Based on cDNA microarray results, integrin-linked kinase (ILK) emerged as an interesting candidate in hypoxia-mediated survival mechanisms employed by cancer cells. This notion was confirmed here by the following observations: the 5' promoter region of the ilk gene contains hypoxia responsive elements (HRE) that bind hypoxia-inducible factor (HIF) transcription factor complexes and drive HRE-luciferase gene expression in reporter assays; ILK protein and kinase activity are induced following hypoxia; downstream targets of ILK signaling are induced following hypoxia treatment; inhibition of ILK leads to increased apoptosis; and HIF and ILK are co-localized within human cancer tissues. The identification of ILK as a player in hypoxia survival signaling employed by cancer cells further validates ILK as a unique target for cancer therapy.