Immune Checkpoint Blockade Delays Cancer and Extends Survival in Murine DNA Polymerase Mutator Syndromes.

Mutations in polymerases Pold1 and Pole exonuclease domains in humans are associated with increased cancer incidence, elevated tumor mutation burden (TMB) and response to immune checkpoint blockade (ICB). Although ICB is approved for treatment of several cancers, not all tumors with elevated TMB respond. Here we generated Pold1 and Pole proofreading mutator mice and show that ICB treatment of mice with high TMB tumors did not improve survival as only a subset of tumors responded. Similarly, introducing the mutator alleles into mice with Kras/p53 lung cancer did not improve survival, however, passaging mutator tumor cells in vitro without immune editing caused rejection in immune-competent hosts, demonstrating the efficiency by which cells with antigenic mutations are eliminated. Finally, ICB treatment of mutator mice earlier, before observable tumors delayed cancer onset, improved survival, and selected for tumors without aneuploidy, suggesting the use of ICB in individuals at high risk for cancer prevention. Highlights: Germline somatic and conditional Pold1 and Pole exonuclease domain mutations in mice produce a mutator phenotype. Spontaneous cancers arise in mutator mice that have genomic features comparable to human tumors with these mutations.ICB treatment of mutator mice with tumors did not improve survival as only a subset of tumors respond. Introduction of the mutator alleles into an autochthonous mouse lung cancer model also did not produce immunogenic tumors, whereas passaging mutator tumor cells in vitro caused immune rejection indicating efficient selection against antigenic mutations in vivo . Prophylactic ICB treatment delayed cancer onset, improved survival, and selected for tumors with no aneuploidy.

RESPIRATION DEFECTS LIMIT SERINE SYNTHESIS REQUIRED FOR LUNG CANCER GROWTH AND SURVIVAL.

Mitochondrial function is important for both energetic and anabolic metabolism. Pathogenic mitochondrial DNA (mtDNA) mutations directly impact these functions, resulting in the detrimental consequences seen in human mitochondrial diseases. The role of pathogenic mtDNA mutations in human cancers is less clear; while pathogenic mtDNA mutations are observed in some cancer types, they are almost absent in others. We report here that the proofreading mutant DNA polymerase gamma ( PolG D256A ) induced a high mtDNA mutation burden in non-small-cell lung cancer (NSCLC), and promoted the accumulation of defective mitochondria, which is responsible for decreased tumor cell proliferation and viability and increased cancer survival. In NSCLC cells, pathogenic mtDNA mutations increased glycolysis and caused dependence on glucose. The glucose dependency sustained mitochondrial energetics but at the cost of a decreased NAD+/NADH ratio that inhibited de novo serine synthesis. Insufficient serine synthesis, in turn, impaired the downstream synthesis of GSH and nucleotides, leading to impaired tumor growth that increased cancer survival. Unlike tumors with intact mitochondrial function, NSCLC with pathogenic mtDNA mutations were sensitive to dietary serine and glycine deprivation. Thus, mitochondrial function in NSCLC is required specifically to sustain sufficient serine synthesis for nucleotide production and redox homeostasis to support tumor growth, explaining why these cancers preserve functional mtDNA. In brief: High mtDNA mutation burden in non-small-cell lung cancer (NSCLC) leads to the accumulation of respiration-defective mitochondria and dependency on glucose and glycolytic metabolism. Defective respiratory metabolism causes a massive accumulation of cytosolic nicotinamide adenine dinucleotide + hydrogen (NADH), which impedes serine synthesis and, thereby, glutathione (GSH) and nucleotide synthesis, leading to impaired tumor growth and increased survival. Highlights: Proofreading mutations in Polymerase gamma led to a high burden of mitochondrial DNA mutations, promoting the accumulation of mitochondria with respiratory defects in NSCLC.Defective respiration led to reduced proliferation and viability of NSCLC cells increasing survival to cancer.Defective respiration caused glucose dependency to fuel elevated glycolysis.Altered glucose metabolism is associated with high NADH that limits serine synthesis, leading to impaired GSH and nucleotide production.Mitochondrial respiration defects sensitize NSCLC to dietary serine/glycine starvation, further increasing survival.

Early Detection of Myeloid-Derived Suppressor Cells in the Lung Pre-Metastatic Niche by Shortwave Infrared Nanoprobes.

Metastatic breast cancer remains a significant source of mortality amongst breast cancer patients and is generally considered incurable in part due to the difficulty in detection of early micro-metastases. The pre-metastatic niche (PMN) is a tissue microenvironment that has undergone changes to support the colonization and growth of circulating tumor cells, a key component of which is the myeloid-derived suppressor cell (MDSC). Therefore, the MDSC has been identified as a potential biomarker for PMN formation, the detection of which would enable clinicians to proactively treat metastases. However, there is currently no technology capable of the in situ detection of MDSCs available in the clinic. Here, we propose the use of shortwave infrared-emitting nanoprobes for the tracking of MDSCs and identification of the PMN. Our rare-earth albumin nanocomposites (ReANCs) are engineered to bind the Gr-1 surface marker of murine MDSCs. When delivered intravenously in murine models of breast cancer with high rates of metastasis, the targeted ReANCs demonstrated an increase in localization to the lungs in comparison to control ReANCs. However, no difference was seen in the model with slower rates of metastasis. This highlights the potential utility of MDSC-targeted nanoprobes to assess PMN development and prognosticate disease progression.

Integrated metabolic and genetic analysis reveals distinct features of human differentiated thyroid cancer.

BACKGROUND: Differentiated thyroid cancer (DTC) affects thousands of lives worldwide each year. Typically, DTC is a treatable disease with a good prognosis. Yet, some patients are subjected to partial or total thyroidectomy and radioiodine therapy to prevent local disease recurrence and metastasis. Unfortunately, thyroidectomy and/or radioiodine therapy often worsen(s) quality of life and might be unnecessary in indolent DTC cases. On the other hand, the lack of biomarkers indicating a potential metastatic thyroid cancer imposes an additional challenge to managing and treating patients with this disease. AIM: The presented clinical setting highlights the unmet need for a precise molecular diagnosis of DTC and potential metastatic disease, which should dictate appropriate therapy. MATERIALS AND METHODS: In this article, we present a differential multi-omics model approach, including metabolomics, genomics, and bioinformatic models, to distinguish normal glands from thyroid tumours. Additionally, we are proposing biomarkers that could indicate potential metastatic diseases in papillary thyroid cancer (PTC), a sub-class of DTC. RESULTS: Normal and tumour thyroid tissue from DTC patients had a distinct yet well-defined metabolic profile with high levels of anabolic metabolites and/or other metabolites associated with the energy maintenance of tumour cells. The consistency of the DTC metabolic profile allowed us to build a bioinformatic classification model capable of clearly distinguishing normal from tumor thyroid tissues, which might help diagnose thyroid cancer. Moreover, based on PTC patient samples, our data suggest that elevated nuclear and mitochondrial DNA mutational burden, intra-tumour heterogeneity, shortened telomere length, and altered metabolic profile reflect the potential for metastatic disease. DISCUSSION: Altogether, this work indicates that a differential and integrated multi-omics approach might improve DTC management, perhaps preventing unnecessary thyroid gland removal and/or radioiodine therapy. CONCLUSIONS: Well-designed, prospective translational clinical trials will ultimately show the value of this integrated multi-omics approach and early diagnosis of DTC and potential metastatic PTC.

Integrated metabolic and genetic analysis reveals distinct features of primary differentiated thyroid cancer and its metastatic potential in humans.

Differentiated thyroid cancer (DTC) affects thousands of lives worldwide every year. Typically, DTC is a treatable disease with a good prognosis. Yet, some patients are subjected to partial or total thyroidectomy and radioiodine therapy to prevent local disease recurrence and metastasis. Unfortunately, thyroidectomy and/or radioiodine therapy often worsen(s) the quality of life and might be unnecessary in indolent DTC cases. This clinical setting highlights the unmet need for a precise molecular diagnosis of DTC, which should dictate appropriate therapy. Here we propose a differential multi-omics model approach to distinguish normal gland from thyroid tumor and to indicate potential metastatic diseases in papillary thyroid cancer (PTC), a sub-class of DTC. Based on PTC patient samples, our data suggest that elevated nuclear and mitochondrial DNA mutational burden, intratumor heterogeneity, shortened telomere length, and altered metabolic profile reflect the potential for metastatic disease. Specifically, normal and tumor thyroid tissues from these patients had a distinct yet well-defined metabolic profile with high levels of anabolic metabolites and/or other metabolites associated with the energy maintenance of tumor cells. Altogether, this work indicates that a differential and integrated multi-omics approach might improve DTC management, perhaps preventing unnecessary thyroid gland removal and/or radioiodine therapy. Well-designed, prospective translational clinical trials will ultimately show the value of this targeted molecular approach. TRANSLATIONAL RELEVANCE: In this article, we propose a new integrated metabolic, genomic, and cytopathologic methods to diagnose Differentiated Thyroid Cancer when the conventional methods failed. Moreover, we suggest metabolic and genomic markers to help predict high-risk Papillary Thyroid Cancer. Both might be important tools to avoid unnecessary surgery and/or radioiodine therapy that can worsen the quality of life of the patients more than living with an indolent Thyroid nodule.

Design and proof of concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain.

Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these "dual-display" phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.

Autophagy Regulates Stress Responses, Metabolism, and Anticancer Immunity.

Autophagy is a catabolic intracellular nutrient-scavenging pathway triggered by nutrient deprivation and stress that captures and degrades intracellular proteins and organelles in lysosomes. The breakdown products are then recycled into metabolic pathways to sustain survival. Organelle turnover by autophagy contributes to quality control and suppresses inflammation. Autophagy is upregulated in many cancers and supports their growth, survival, and malignancy in a tumor cell-autonomous fashion. Host autophagy also promotes tumor growth by maintaining a supply of essential nutrients and suppressing innate and adaptive antitumor immune responses. Autophagy is also upregulated in response to cancer therapy and confers treatment resistance. Thus, autophagy is a cancer vulnerability and its inhibition is under investigation as a novel therapeutic approach.

Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain.

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SIGNIFICANCE: The ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

B7 immune-checkpoints as targets for the treatment of neuroendocrine tumors.

The B7 family, and their receptors, the CD28 family, are major immune checkpoints that regulate T-cell activation and function. In the present study, we explore the role of two B7 immune-checkpoints: HERV-H LTR-Associating Protein 2 (HHLA2) and B7 Family Member, H4 (B7x), in the progression of gastrointestinal and pancreatic neuroendocrine tumors (GINETs and PNETs). We demonstrated that both HHLA2 and B7x were expressed to a high degree in human GINETs and PNETs. We determined that the expression of B7x and HHLA2 correlates with higher grade and higher incidence of nodal and distant spread. Furthermore, we confirmed that HIF-1α overexpression is associated with the upregulation of B7x both in our in vivo (animal model) and in vitro (cell culture) models. When grown in vitro, islet tumor ß-cells lack B7x expression, unless cultured under hypoxic conditions, which results in both hypoxia-inducible factor 1 subunit alpha (HIF-1α) and B7x upregulation. In vivo, we demonstrated that Men1/B7x double knockout (KO) mice (with loss of B7x expression) exhibited decreased islet ß-cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single knockout mice. We have also shown that systemic administration of a B7x mAb to our Men1 KO mice with PNETs promotes an antitumor response mediated by increased T-cell infiltration. These findings suggest that B7x may be a critical mediator of tumor immunity in the tumor microenvironment of NETs. Therefore, targeting B7x offers an attractive strategy for the immunotherapy of patients suffering from NETs.

Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response.

Macroautophagy (hereafter autophagy) degrades and recycles intracellular components to sustain metabolism and survival during starvation. Host autophagy promotes tumor growth by providing essential tumor nutrients. Autophagy also regulates immune cell homeostasis and function and suppresses inflammation. Although host autophagy does not promote a T-cell anti-tumor immune response in tumors with low tumor mutational burden (TMB), whether this was the case in tumors with high TMB was not known. Here we show that autophagy, especially in the liver, promotes tumor immune tolerance by enabling regulatory T-cell function and limiting stimulator of interferon genes, T-cell response and interferon-γ, which enables growth of high-TMB tumors. We have designated this as hepatic autophagy immune tolerance. Autophagy thereby promotes tumor growth through both metabolic and immune mechanisms depending on mutational load and autophagy inhibition is an effective means to promote an antitumor T-cell response in high-TMB tumors.

Phase I neoadjuvant study of intravesical recombinant fowlpox-GM-CSF (rF-GM-CSF) or fowlpox-TRICOM (rF-TRICOM) in patients with bladder carcinoma.

Intravesical BCG is a highly effective treatment for high-grade nonmuscle invasive bladder cancer and carcinoma in situ (CIS); however, for patients who are either resistant or become unresponsive to BCG therapy there is a need for alternative treatment approaches. This study examined the safety and feasibility of intravesically administered recombinant fowlpox virus encoding GM-CSF (Arm A) or TRICOM (Arm B); and the local and systemic immunologic responses generated to the vector(s). Twenty bladder cancer patients scheduled for cystectomy as their standard of care received preoperatively four weekly doses of intravesical recombinant fowlpox. Treatment was well tolerated, however, three patients experienced transient elevations of liver transaminases, with one rising to the level of a DLT. Cystectomy derived tumor and normal bladder mucosa demonstrated mRNA for the virally encoded LacZ gene supporting effective infection/transfection. Detected serum antibody to the LacZ encoding ß-galactosidase indicated successful expression of vector-encoding gene products and the ability to immunize via the bladder site. H&E and IHC using a panel of immune cell specific antigens demonstrated immune cell infiltration of the bladder wall. These findings demonstrate good safety profile, successful infection/transfection, ability to generate systemic immune response, and local recruitment of immune cell populations with intravesical administration of fowlpox-based constructs encoding for GM-CSF(rF-GM-CSF) or TRICOM (rF-TRICOM), and support further evaluation of this treatment modality for bladder cancer.

Non-oncogenic Acute Viral Infections Disrupt Anti-cancer Responses and Lead to Accelerated Cancer-Specific Host Death.

In light of increased cancer prevalence and cancer-specific deaths in patients with infections, we investigated whether infections alter anti-tumor immune responses. We report that acute influenza infection of the lung promotes distal melanoma growth in the dermis and leads to accelerated cancer-specific host death. Furthermore, we show that during influenza infection, anti-melanoma CD8+ T cells are shunted from the tumor to the infection site, where they express high levels of the inhibitory receptor programmed cell death protein 1 (PD-1). Immunotherapy to block PD-1 reverses this loss of anti-tumor CD8+ T cells from the tumor and decreases infection-induced tumor growth. Our findings show that acute non-oncogenic infection can promote cancer growth, raising concerns regarding acute viral illness sequelae. They also suggest an unexpected role for PD-1 blockade in cancer immunotherapy and provide insight into the immune response when faced with concomitant challenges.

Targeting tumor-associated macrophages to combat pancreatic cancer.

The tumor microenvironment is replete with cells that evolve with and provide support to tumor cells during the transition to malignancy. The hijacking of the immune system in the pancreatic tumor microenvironment is suggested to contribute to the failure to date to produce significant improvements in pancreatic cancer survival by various chemotherapeutics. Regulatory T cells, myeloid derived suppressor cells, and fibroblasts, all of which constitute a complex ecology microenvironment, can suppress CD8+ T cells and NK cells, thus inhibiting effector immune responses. Tumor-associated macrophages (TAM) are versatile immune cells that can express different functional programs in response to stimuli in tumor microenvironment at different stages of pancreatic cancer development. TAM have been implicated in suppression of anti-tumorigenic immune responses, promotion of cancer cell proliferation, stimulation of tumor angiogenesis and extracellular matrix breakdown, and subsequent enhancement of tumor invasion and metastasis. Many emerging agents that have demonstrated efficacy in combating other types of tumors via modulation of macrophages in tumor microenvironments are, however, only marginally studied for pancreatic cancer prevention and treatment. A better understanding of the paradoxical roles of TAM in pancreatic cancer may pave the way to novel preventive and therapeutic approaches. Here we give an overview of the recruitment and differentiation of macrophages, TAM and pancreatic cancer progression and prognosis, as well as the potential preventive and therapeutic targets that interact with TAM for pancreatic cancer prevention and treatment.

Recombinant Poxvirus and the Tumor Microenvironment: Oncolysis, Immune Regulation and Immunization.

Oncolytic viruses (OVs) are being extensively studied for their potential roles in the development of cancer therapy regimens. In addition to their direct lytic effects, OVs can initiate and drive systemic antitumor immunity indirectly via release of tumor antigen, as well as by encoding and delivering immunostimulatory molecules. This combination makes them an effective platform for the development of immunotherapeutic strategies beyond their primary lytic function. Engineering the viruses to also express tumor-associated antigens (TAAs) allows them to simultaneously serve as therapeutic vaccines, targeting and amplifying an immune response to TAAs. Our group and others have shown that vaccinating intratumorally with a poxvirus that encodes TAAs, in addition to immune stimulatory molecules, can modulate the tumor microenvironment, overcome immune inhibitory pathways, and drive both local and systemic tumor specific immune responses.

A national multicenter phase 2 study of prostate-specific antigen (PSA) pox virus vaccine with sequential androgen ablation therapy in patients with PSA progression: ECOG 9802.

BACKGROUND: E9802 was a phase 2 multi-institution study conducted to evaluate the safety and effectiveness of vaccinia and fowlpox prostate-specific antigen (PSA) vaccine (step 1) followed by combination with androgen ablation therapy (step 2) in patients with PSA progression without visible metastasis. OBJECTIVE: To test the hypothesis that vaccine therapy in this early disease setting will be safe and have a biochemical effect that would support future studies of immunotherapy in patients with minimal disease burden. DESIGN, SETTING, AND PARTICIPANTS: Patients who had PSA progression following local therapy were treated with PROSTVAC-V (vaccinia)/TRICOM on cycle 1 followed by PROSTVAC-F (fowlpox)/TRICOM for subsequent cycles in combination with granulocyte-macrophage colony-stimulating factor (step 1). Androgen ablation was added on progression (step 2). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Step 1 primary end points included progression at 6 mo and characterization of change in PSA velocity pretreatment to post-treatment. Step 2 end points included PSA response with combined vaccine and androgen ablation. RESULTS AND LIMITATIONS: In step 1, 25 of 40 eligible patients (63%) were progression free at 6 mo after registration (90% confidence interval [CI], 48-75). The median pretreatment PSA velocity was 0.13 log(PSA)/mo, in contrast to median postregistration velocity of 0.09 log(PSA)/mo (p=0.02), which is an increase in median PSA doubling time from 5.3 mo to 7.7 mo. No grade ≥4 treatment-related toxicity was observed. In the 27 patients eligible and treated for step 2, 20 patients achieved a complete response (CR) at 7 mo (CR rate: 74%; 90% CI, 57-87). Although supportive of larger studies in the cooperative group setting, this study is limited by the small number of patients and the absence of a control group as in a phase 3 study. CONCLUSIONS: A viral PSA vaccine can be administered safely in the multi-institutional cooperative group setting to patients with minimal disease volume alone and combined with androgen ablation, supporting the feasibility of future phase 3 studies in this population. PATIENT SUMMARY: These data support consideration of vaccine therapy earlier in the course of prostate cancer progression with minimal disease burden in future studies of vaccine approaches in earlier stages of disease.

PLGA-polymer encapsulating tumor antigen and CpG DNA administered into the tumor microenvironment elicits a systemic antigen-specific IFN-γ response and enhances survival.

Critical to the generation of an effective therapeutic antitumor immune response is the elicitation of effective antigen presentation coupled with overcoming tumor-immune escape mechanisms. Towards this end, we aimed to understand the therapeutic effectiveness of a polymer based vaccine approach at enhancing the anti-tumor responses in a tumor-bearing mouse model. While we and others have previously demonstrated the effectiveness of PLGA based systems in delivering antigen etc., studies scarcely focus on understanding the immunological mechanisms of polymer based therapies in tumor bearing treatment models. Considering tumors modulate the immune system and consequently the efficacy of therapies, understanding treatment mechanisms in the presence of tumor will help lead to more efficacious treatment options. We demonstrate here that a poly(lactic-co-glycolic acid) (PLGA) based delivery system encapsulating tumor antigen (OVA) and the TLR9 agonist CpG motif DNA administered into the tumor microenvironment initiates an effective type 1 mediated (IFN-γ producing) anti-tumor response in a syngeneic murine model of T cell lymphoma (E.G7-OVA). Although E.G7-OVA tumors spontaneously generate antigen specific CTLs in draining lymph nodes (LN), tumors progress rapidly. Modulation of the tumor microenvironment via local PLGA based therapy led to the generation of a systemic antigen specific Th1 response, absent in the non-polymer delivery method, subsequently associated with reduced tumor growth and prolongation of survival. These studies provide further insight into the use of a PLGA-based therapeutic approach at modulating the tumor microenvironment and highlight the need for analyzing the treatment effects in a tumor bearing model.

Vaccinia virus leads to ATG12­ATG3 conjugation and deficiency in autophagosome formation.

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12­ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

ANALYSIS OF DENDRITIC CELL STIMULATION UTILIZING A MULTI-FACETED NANOPOLYMER DELIVERY SYSTEM AND THE IMMUNE MODULATOR 1-METHYL TRYPTOPHAN.

Dendritic cells (DCs) play a pivotal role in immune modulation. Therefore, understanding and regulating the mechanism of DC activation is paramount for functional optimization of any immunotherapy strategy. In particular, the paradoxical ability of DCs to secrete the immune suppressive enzyme indoleamine 2, 3-dioxygenase (IDO) and the suppressive cytokine IL-10 during the course of, and in response to, stimulation is of great interest. 1-Methyl-Tryptophan (1 MT) is a known inhibitor of IDO and has thus been administered in numerous in vitro and in vivo systems to block IDO activity. However, the effect 1 MT has on DCs beyond inhibiting IDO, especially in therapeutic models, has rarely been analyzed. In the current study, we have administered 1 MT via a nanopolymer-based delivery system in conjunction with an antigen (ovalbumin, OVA) and an adjuvant (CpG motif DNA) to determine both the effects of 1 MT on DCs and the resulting efficacy of the polymer-based treatments. 1 MT delivery alone, either via the polymer-based delivery vehicle or dissolved in solution, induced no significant change in DC activation as measured by surface expression of CD80, CD86, and MHCII and several secreted products such as IL-12. These same factors were upregulated however, when 1 MT was delivered in conjunction with OVA and CpG. Although soluble delivery of these components increased the levels of expression and secretion of key proteins, a differential effect of DC stimulation was seen as a result of the polymer delivery system. The T cell suppressive IL-10 secretion was lower with the polymer-based treatments and IL-12 immune-enhancing secretion was increased when 1 MT was supplemented into the polymer system. As a result, including 1 MT in the polymers along with OVA and CpG was seen to have additional effects on DC stimulation and was able to shift DCs to a state more indicative of inducing a Th1-type response.