Immune Stimulatory Capacity Research Articles

Xenogeneic equine stem cells activate anti-tumor adaptive immunity in a 4T1-based intraductal mouse model for triple-negative breast cancer: proof-of-principle.

Triple-negative breast cancer (TNBC) remains difficult to treat, especially due to ineffective immune responses. Current treatments mainly aim at a cytotoxic effect, whereas (stem) cell therapies are being investigated for their immune stimulatory capacities to initiate the anti-tumor immunity. Here, a thoroughly characterized, homogenous and non-tumorigenic mixture of equine mesenchymal stem cells (eMSCs) harvested from horse peripheral blood as innovative xenogeneic immunomodulators were tested in a 4T1-based intraductal mouse model for TNBC. The eMSCs significantly reduced 4T1 progression upon systemic injection, with induction of inflammatory mediators and T-cell influx in primary tumors, already after a single dose. These xenogeneic anti-cancer effects were not restricted to MSCs as systemic treatment with alternative equine epithelial stem cells (eEpSCs) mimicked the reported disease reduction. Mechanistically, effective eMSC treatment did not rely on the spleen as systemic entrapment site, whereas CD4+ and CD8α+ T-cell infiltration and activation were critical. These results show that eMSCs and potentially also other equine stem cell types can be a valuable TNBC treatment strategy for further (pre)clinical evaluation.

Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens.

House dust mite (HDM) is a major cause of respiratory allergic diseases. Dendritic cells (DCs) play a central role in orchestrating adaptive allergic immune responses. However, it remains unclear how DCs become activated by HDM. Biochemical functions of the major HDM allergens Der p 1 (cysteine protease) and Der p 2 (MD2-mimick) have been implicated to contribute to DC activation. We investigated the immune activating potential of HDM extract and its major allergens Der p 1 and Der p 2 using monocyte-derived DCs (moDCs). Maturation and activation markers were monitored by flow cytometry and cytokine production by ELISA. Allergen depletion and proteinase K digestion were used to investigate the involvement of proteins, and in particular of the major allergens. Inhibitors of spleen tyrosine kinase (Syk), Toll-like receptor 4 (TLR4) and of C-type lectin receptors (CLRs) were used to identify the involved receptors. The contribution of endotoxins in moDC activation was assessed by their removal from HDM extract. HDM extract induced DC maturation and cytokine responses in contrast to the natural purified major allergens Der p 1 and Der p 2. Proteinase K digestion and removal of Der p 1 or Der p 2 did not alter the immune stimulatory capacity of HDM extract. Antibodies against the CLRs Dectin-1, Dectin-2, and DC-SIGN did not affect cytokine responses. In contrast, Syk inhibition partially reduced IL-6, IL-12 and completely blocked IL-10. Blocking TLR4 signaling reduced the HDM-induced IL-10 and IL-12p70 induction, but not IL-6, while endotoxin removal potently abolished the induced cytokine response. Our data strongly suggest that HDM-induced DC activation is neither dependent on Der p 1 nor Der p 2, but depend on Syk and TLR4 activation, which might suggest a crosstalk between Syk and TLR4 pathways. Our data highlight that endotoxins play a potent role in immune responses targeting HDM.

Chemically programmed STING-activating nano-liposomal vesicles improve anticancer immunity

The often immune-suppressive tumor microenvironment (TME) may hinder immune evasion and response to checkpoint blockade therapies. Pharmacological activation of the STING pathway does create an immunologically hot TME, however, systemic delivery might lead to undesired off-target inflammatory responses. Here, we generate a small panel of esterase-activatable pro-drugs based on the structure of the non-nucleotide STING agonist MSA-2 that are subsequently stably incorporated into a liposomal vesicle for intravenous administration. The pharmacokinetic properties and immune stimulatory capacity of pro-drugs delivered via liposomes (SAProsomes) are enhanced compared to the free drug form. By performing efficacy screening among the SAProsomes incorporating different pro-drugs in syngeneic mouse tumor models, we find that superior therapeutic performance relies on improved delivery to the desired tumor and lymphoid compartments. The best candidate, SAProsome-3, highly stimulates secretion of inflammatory cytokines and creates a tumoricidal immune landscape. Notably, upon application to breast cancer or melanoma mouse models, SAProsome-3 elicits durable remission of established tumors and postsurgical tumor-free survival while decreasing metastatic burden without significant systemic toxicity. In summary, our work establishes the proof of principle for a better targeted and more efficient and safe STING agonist therapy.

Skewing of UV-induced cell death pathways using TLR4 ligands

Abstract Toll-like receptor 4 (TLR4) is a canonical innate immune receptor for the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). TLR4 deficiency has been shown to contribute to UV-induced carcinogenesis in epidermal cells. UV-induced cellular damage results in apoptosis which is characterized by a non-inflammatory cell death through the cleavage and activation of caspase-3. Utilizing bacterial enzyme combinational chemistry (BECC) we generated hypoacylated lipid A moieties from Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium which have moderately attenuated immune-stimulatory capacity. Continuously exposing TLR4 to these innate immune ligands leads to tolerization and decreased expression of the receptor in mammalian cells. We hypothesize that skewing cell death away from apoptosis using lipid A mimetics to lower TLR4 expression prior to UV exposure will reduce carcinogenic potential. To analyze the roll of TLR4 in UV-induced carcinogenesis, wildtype and TLR4-deficient bone marrow-derived macrophage (BMDM) cell lines were tolerized with hexa-acylated or penta-acylated lipid A from multiple Gram-negative species and exposed to either 10 or 25 mJ/cm 2doses of UVB radiation. UV-irradiated BMDM cells show evidence of apoptosis including cleavage of DNA creating a ladder, whereas UV-irradiated BMDM cells tolerized with lipid A mimetics show a lack of apoptosis. The data suggests that tolerization of TLR4 using canonical ligands may be a useful tool in promoting DNA repair and maintaining immune responses following UV-induced damage. The research was supported by a Fisher College of Science and Mathematics Undergraduate Research Grant.

Radiation to all macroscopic sites of tumor permits greater systemic antitumor response to in situ vaccination

BackgroundThe antitumor effects of external beam radiation therapy (EBRT) are mediated, in part, by an immune response. We have reported that a single fraction of 12 Gy EBRT combined with...

Abstract 697: Defining anti-tumor immune stimulatory mechanisms of MEM-288, a CD40 ligand and IFN-beta dual-transgene armed oncolytic adenovirus

Abstract MEM-288 is a dual-transgene armed oncolytic adenovirus being developed for cancer treatment as a standalone agent or in combination with immune checkpoint inhibitors (ICI). MEM-288 is armed with two potent immune agonist transgenes: MEM40, a novel chimeric CD40 ligand (CD40L) designed for stable cell surface expression of CD40L that functions as a potent activator of CD40 expressing dendritic cells (DCs), and the DC and T cell activating cytokine IFNβ. We have evaluated MEM-288 activity in multiple in vivo immunocompetent mouse models to determine impact on systemic antitumor T cell activity after intratumoral virus injection. We were able to readily detect both tumor and immune cell infection and transgene expression following MEM-288 intratumoral administration in mouse tumor models and freshly resected human NSCLC tumors. MEM-288 injection in the syngeneic mouse lung metastatic model 344SQ demonstrated anti-metastatic activity that correlated with a significant enhancement in tumor infiltrating lymphocytes (TILs) and an increase in the systemic antitumor CD8 T cell response. MEM-288 also generated a highly efficacious response against both injected and distant non-injected contralateral tumors in the B16-F10 melanoma model, both as a monotherapy and in combination with ICI. These antitumor and immune responses were only found with MEM-288 as compared to the base adenovirus vector encoding only GFP transgene, indicating the observed effects were highly transgene-dependent. Dissecting the immune stimulatory effects further, we found MEM-288 injection in B16 tumors led to a significant increase in DC activation markers CD80 and CD86 suggesting that DCs may represent a key target of MEM-288 encoded transgenes leading to systemic T cell responses. In support of this hypothesis, we found the MEM-288 CD8 T cell response was completely abolished in BATF3 knockout (KO) mice which lack the cDC1 DC subset (CD8α conventional DC deficient). This suggests that cDC1s are a key mediator of MEM-288 activity. Furthermore, MEM-288 activity was also impaired in both CD40 and type 1 IFN receptor (IFNAR1) KO mice suggesting that both transgenes mediate MEM-288 immune stimulation. In summary, these results demonstrate MEM-288's capacity to induce a potent increase in systemic T cell responses through transgene-mediated stimulation of tumor DCs. This immune stimulatory capacity complements the additional mechanistic features of MEM-288, including enhanced tumor-specific viral replication, oncolysis, and tumor antigen release. MEM-288 is currently being prepared for first-in-human clinical testing in solid tumors as a monotherapy and in combination with ICI. Citation Format: Hong Zheng, Chase D. Powell, Scott Antonia, Mark J. Cantwell, Bradford A. Perez, Amer A. Beg. Defining anti-tumor immune stimulatory mechanisms of MEM-288, a CD40 ligand and IFN-beta dual-transgene armed oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 697.

Adoptive T Cell Therapy Is Complemented by Oncolytic Virotherapy with Fusogenic VSV-NDV in Combination Treatment of Murine Melanoma.

Simple SummaryCancer immunotherapy involves the application of strategies aimed at enhancing the body’s immune system to recognize and clear tumor cells. One such immunotherapeutic approach utilizes cytotoxic immune cells (T cells) harvested from the patient, which are expanded and activated, followed by re-infusion to the same patient. This process is termed adoptive cell therapy (ACT). Although effective in a limited setting, efforts are being made to improve these therapies through the development of rationally designed combination treatments. We have developed an approach, whereby tumors are pretreated with a virus, which has oncolytic effects on the tumor cells, in addition to modulating changes in the tumor microenvironment, thereby improving the recruitment of the adoptively transferred cytotoxic T cells and resulting in synergistic therapeutic responses in the tumor. This results in a substantial prolongation of survival, as demonstrated in an immune-competent mouse model of melanoma.Cancer immunotherapies have made major advancements in recent years and are becoming the prevalent treatment options for numerous tumor entities. However, substantial response rates have only been observed in specific subsets of patients since pre-existing factors determine the susceptibility of a tumor to these therapies. The development of approaches that can actively induce an anti-tumor immune response, such as adoptive cell transfer and oncolytic virotherapy, have shown clinical success in the treatment of leukemia and melanoma, respectively. Based on the immune-stimulatory capacity of oncolytic VSV-NDV virotherapy, we envisioned a combination approach to synergize with adoptive T cell transfer, in order to enhance tumor cell killing. Using the immune-competent B16 melanoma model, we demonstrate that combination treatment has beneficial effects on the suppressive microenvironment through upregulation of MHC-I and maintaining low expression levels of PD-L1 on tumor cells. The approach led to additive cytotoxic effects and improved the recruitment of T cells to virus-infected tumor cells in vitro and in vivo. We observed substantial delays in tumor growth and evidence of abscopal effects, as well as prolongation of overall survival time when administered at clinically relevant dosing conditions. Our results indicate that treatment with oncolytic VSV-NDV, combined with adoptive T cell therapy, induces multi-mechanistic and synergistic tumor responses, which supports the further development of this promising translational approach.

Liposomes derived from Mycobacterium smegmatis promote immune activation of mice bone marrow-derived dendritic cells.

Tuberculosis (TB) is the leading cause of mortality due to infectious diseases. The development of new generation vaccines against TB is of paramount importance for the control of the disease. In previous studies, liposomes obtained from lipids of Mycobacterium smegmatis (LMs) demonstrated their immunogenicity and protective capacity against Mycobacterium tuberculosis in mice. To characterize the immunomodulatory capacity of this experimental vaccine candidate, in the current study, the stimulatory capacity of LMs was determined on bone marrow-derived dendritic cells (BMDCs) from mice. LMs were obtained and incubated with mature BMDCs. The internalization of LMs by BMDCs was studied by confocal microscopy, and the LMs immune-stimulatory capacity was determined by the expression of surface molecules (CD86 and MHCII) and the cytokine production (interleukin [IL]-12, interferon-Υ, tumor necrosis factor-α, and IL-10) 24 h after exposure to LMs. The interaction of LMs with BMDCs and its internalization was demonstrated as well as the immune activation of BMDCs, characterized by the increased expression of CD86 and the production of IL-12. The LMs internalization and immune activation of BMDCs were blocked in the presence of cytochalasin, filipin III and chlorpromazine, which demonstrated that internalization of LMs by BMDCs is a key process for the LMs induced immune activation of BMDCs. The results obtained support the further evaluation of LMs as a mycobacterial vaccine, adjuvant, and in immunotherapy.

The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1β (IL-1β) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1β production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

4O - Novel dendritic cell based immunotherapy for advanced cancer

BackgroundImmune-checkpoint inhibitors (ICI) such as Ipilumimab and Nivolumab have made long-term survival a real possibility for advanced cancer patients. Despite the success, only a proportion of patients receiving ICI show meaningful clinical benefit. Developing a more personalized treatment strategy using patient’s own immune cells may be a promising approach for treating non-responding patients. Natural circulating dendritic cells (nDC) such as CD1c+ DC (cDC2) subset and plasmacytoid DC (pDC) are essential for efficient activation of immune responses after ICI therapy. Thus understanding the condition of these cells in patients and devising new strategies to improve their functions may help improve response to ICI in advanced cancer patients. MethodsTo investigate this we measured the number and phenotype of nDC in 29 advanced cancer patients versus 21 age and gender-matched healthy controls using flow cytometry. We further identified that inhibition of the p38 MAPK pathway using small molecule inhibitors in cDC2 cells increases their immune-stimulatory capacity, measured by functional assays. ResultsThe number of pDC were significantly reduced in cancer patients as compared to healthy controls (1221 vs 2843 pDC/mL) (p-value<0.01). Although the number of cDC2 were slightly reduced in cancer patients (4476 vs 5053 of cDC2/mL), a good proportion of patients had sufficient number of DC eligible for a DC-based immunotherapy. Patient cDC2 were immune-suppressed with low secretion of immune-stimulatory cytokines (IL-12) and impaired homing to lymph-nodes. We identified that the p38 MAPK signalling pathway was controlling immune-suppression in cDC2 cells. Inhibiting this pathway restored the secretion of IL-12 in all patients (n=5), increased the homing of DC to lymph-node chemokines, and prevented them from secreting the immune-suppressive cytokine, IL-10. ConclusionsThe number and phenotype of nDC are suppressed in advanced cancer patients. Inhibition of p38 MAPK can restore the function of patient cDC2 subset. Co-culture of these p38-inhibited cDC2 with pDC further show promising results important for developing advanced DC vaccines. Our study has paved the way for a phase I clinical trial of adoptive transfer of p38-MAPK inhibited cDC2 in 24 advanced cancer patients. Legal entity responsible for the studyUniversity of Nottingham. FundingUniversity of Nottingham. DisclosureAll authors have declared no conflicts of interest.

Abstract 115: A CD40 agonist potentiates the efficacy and immune-stimulatory capacity of chemotherapy in combination with a focal adhesion kinase inhibitor in a mouse model of pancreatic ductal adenocarcinoma

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) has proved to be remarkably resistant to immune based therapies. This resistance may be in part due to the PDAC microenvironment that is characterized by an abundance of myeloid cells, a scarcity of effector T cells and a dense fibrotic stroma. Activation of focal adhesion kinase (FAK) signaling promotes the formation of an immune suppressive microenvironment in PDAC, and FAK inhibition (FAKi) can reduce stromal density and improve the sensitivity of tumors to chemotherapy. Likewise, CD40-dependent innate immune responses can disrupt the stromal matrix that surrounds the tumor and sensitize tumors to the cytotoxic and immune-stimulatory effects of chemotherapy. Here, we hypothesize that FAK signaling may limit the efficacy of CD40 agonists and thus, FAK and CD40 may be non-redundant pathways that can be targeted for improving outcomes to cytotoxic chemotherapy. METHODS: For our studies, we used polyclonal pancreatic cancer cells derived from the KrasG21D/+; Trp53R172H/+; Pdx-1 Cre (KPC) genetic mouse model of PDAC, which recapitulates many of the features of human disease, including a paucity of T cells and a robust infiltration of myeloid cells. KPC cell lines were subcutaneously implanted into syngeneic C57BL/6 mice. Approximately 2 weeks after tumor implantation, mice were block randomized to receive treatment with a CD40 agonist (FGK45) or PBS administered prior to (5 days prior) or after (2 days post) gemcitabine (gem) chemotherapy with or without FAKi. RESULTS: We first examined the impact of chemotherapy in combination with FAKi to enhance the immune stimulatory capacity of a CD40 agonist. We found that the triple combination (FAKi+gem→FGK45), in which FGK45 is given after FAKi and gem, significantly reduced tumor growth, increased tumor necrosis, and improved survival compared to FAKi alone or gem→FGK45. In this treatment setting, both gem→FGK45 and the triple combination significantly increased the CD8+/Foxp3+ ratio and the proliferation of CD8+ T cells in tumors. We next examined the capacity of a CD40 agonist to enhance the efficacy of chemotherapy, with or without FAKi. The triple combination (FGK45→FAKi+gem), in which FGK45 is given before FAKi and gem, significantly inhibited tumor growth and improved survival compared to all other groups. In this treatment setting, both FGK45→FAKi+gem and FGK45→gem increased the number and proliferation of CD3+ T cells in tumors. Importantly, the triple combination significantly increased the CD8+/Foxp3+ ratio compared to FGK45→Gem, which may account in part for the improvement in tumor response. CONCLUSION: FAK and CD40 signaling are non-redundant pathways that can be targeted to reverse immune suppressive features in the tumor microenvironment and improve PDAC sensitivity to chemotherapy. Citation Format: Meredith L. Stone, Kathleen Graham, Daniel Aldridge, Kanika Jain, Xiaoqing Pan, Jonathan Pachter, Gregory Beatty. A CD40 agonist potentiates the efficacy and immune-stimulatory capacity of chemotherapy in combination with a focal adhesion kinase inhibitor in a mouse model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 115.

Engineered Salmonella enterica serovar Typhimurium overcomes limitations of anti-bacterial immunity in bacteria-mediated tumor therapy

ABSTRACT Cancer is one of the leading causes of death in the industrialized world and represents a tremendous social and economic burden. As conventional therapies fail to provide a sustainable cure for most cancer patients, the emerging unique immune therapeutic approach of bacteria-mediated tumor therapy (BMTT) is marching towards a feasible solution. Although promising results have been obtained with BMTT using various preclinical tumor models, for advancement a major concern is immunity against the bacterial vector itself. Pre-exposure to the therapeutic agent under field conditions is a reasonable expectation and may limit the therapeutic efficacy of BMTT. In the present study, we investigated the therapeutic potential of Salmonella and E. coli vector strains in naïve and immunized tumor bearing mice. Pre-exposure to the therapeutic agent caused a significant aberrant phenotype of the microenvironment of colonized tumors and limited the in vivo efficacy of established BMTT vector strains Salmonella SL7207 and E. coli Symbioflor-2. Using targeted genetic engineering, we generated the optimized auxotrophic Salmonella vector strain SF200 (ΔlpxR9 ΔpagL7 ΔpagP8 ΔaroA ΔydiV ΔfliF) harboring modifications in Lipid A and flagella synthesis. This combination of mutations resulted in an increased immune-stimulatory capacity and as such the strain was able to overcome the efficacy-limiting effects of pre-exposure. Thus, we conclude that any limitations of BMTT concerning anti-bacterial immunity may be countered by strategies that optimize the immune-stimulatory capacity of the attenuated vector strains.

Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo

Allergies to weed pollen including members of the Compositae family, such as mugwort, ragweed, and feverfew are spreading worldwide. To efficiently treat these newly arising allergies, allergen specific immunotherapy needs to be improved. Therefore, we generated novel vaccine candidates consisting of the TLR5-ligand Flagellin A from Listeria and the major mugwort allergen Art v 1 including either the wild type Art v 1 sequence (rFlaA:Artv1) or a hypoallergenic variant (rFlaA:Artv1hyp) with reduced IgE-binding capacity. Immune modulating capacity of these constructs and respective controls was evaluated in vitro and in vivo. Incorporation of hypoallergenic Art v 1 derivative did not interfere with the resulting fusion proteins’ immune stimulatory capacity. Both rFlaA:Artv1 and rFlaA:Artv1hyp induced a prominent, mTOR-dependent, IL-10 secretion from murine dendritic cells, and suppressed allergen-specific TH2-cytokine secretion in vitro and in vivo. Both conjugates retained the capacity to induce rFlaA-specific antibody responses while efficiently inducing production of Art v 1-specific IgG1 and IgG2a antibodies in mice. Interestingly, only the suppression of TH2-cytokine secretion by rFlaA:Artv1 (but not rFlaA:Artv1hyp) was paralleled by a strong secretion of IFN-γ. In summary, we provided evidence that incorporating hypoallergens into flagellin:allergen fusion proteins is a suitable strategy to further improve these promising vaccine candidates.

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression.

Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

Intra-Peritoneal Administration of Mitochondrial DNA Provokes Acute Lung Injury and Systemic Inflammation via Toll-Like Receptor 9.

The pathogenesis of sepsis is complex. Mitochondrial dysfunction, which is responsible for energy metabolism, intrinsic apoptotic pathway, oxidative stress, and systemic inflammatory responses, is closely related with severe sepsis induced death. Mitochondria DNA (mtDNA) contain un-methylated cytosine phosphate guanine (CpG) motifs, which exhibit immune stimulatory capacities. The aim of this study was to investigate the role and mechanism of mtDNA release on lipopolysaccharide (LPS) induced acute lung injury (ALI) and systemic inflammation. Following LPS injection, plasma mtDNA copies peak at 8 h. Compared with wild-type (WT) mice, mtDNA in toll like receptor 4 knockout (TLR4 KO) mice were significantly decreased. MtDNA intra-peritoneal administration causes apparent ALI as demonstrated by increased lung injury score, bronchoalveolar lavage fluid (BALF) total protein and wet/dry (W/D) ratio; mtDNA injection also directly provokes systemic inflammation, as demonstrated by increased IL-1β, IL-6, high-mobility group protein B1 (HMGB1) level; while nuclear DNA (nDNA) could not induce apparent ALI and systemic inflammation. However, compared with WT mice, TLR4 KO could not protect from mtDNA induced ALI and systemic inflammation. Specific TLR9 inhibitor, ODN 2088 pretreatment can significantly attenuate mtDNA induced ALI and systemic inflammation, as demonstrated by improved lung injury score, decreased lung wet/dry ratio, BALF total protein concentration, and decreased systemic level of IL-1β, IL-6 and HMGB1. MtDNA administration activates the expression of p-P38 mitogen-activated protein kinases (MAPK) in lung tissue and specific TLR9 inhibitor pretreatment can attenuate this activation. Thus, LPS-induced mtDNA release occurs in a TLR4-dependent manner, and mtDNA causes acute lung injury and systemic inflammation in a TLR9-dependent and TLR4-independent manner.

TNFRSF14 aberrations in follicular lymphoma increase clinically significant allogeneic T-cell responses

AbstractDonor T-cell immune responses can eradicate lymphomas after allogeneic hematopoietic stem cell transplantation (AHSCT), but can also damage healthy tissues resulting in harmful graft-versus-host disease (GVHD). Next-generation sequencing has recently identified many new genetic lesions in follicular lymphoma (FL). One such gene, tumor necrosis factor receptor superfamily 14 (TNFRSF14), abnormal in 40% of FL patients, encodes the herpes virus entry mediator (HVEM) which limits T-cell activation via ligation of the B- and T-lymphocyte attenuator. As lymphoma B cells can act as antigen-presenting cells, we hypothesized that TNFRSF14 aberrations that reduce HVEM expression could alter the capacity of FL B cells to stimulate allogeneic T-cell responses and impact the outcome of AHSCT. In an in vitro model of alloreactivity, human lymphoma B cells with TNFRSF14 aberrations had reduced HVEM expression and greater alloantigen-presenting capacity than wild-type lymphoma B cells. The increased immune-stimulatory capacity of lymphoma B cells with TNFRSF14 aberrations had clinical relevance, associating with higher incidence of acute GVHD in patients undergoing AHSCT. FL patients with TNFRSF14 aberrations may benefit from more aggressive immunosuppression to reduce harmful GVHD after transplantation. Importantly, this study is the first to demonstrate the impact of an acquired genetic lesion on the capacity of tumor cells to stimulate allogeneic T-cell immune responses which may have wider consequences for adoptive immunotherapy strategies.

Tu1381 Gluten-Free Crops Can Contain Proteins With Innate Immune Stimulatory Capacity: Influence of Environmental Factors

Tu1381 Gluten-Free Crops Can Contain Proteins With Innate Immune Stimulatory Capacity: Influence of Environmental Factors

Abstract A170: The mode of action of Orf virus – a novel viral vector for therapeutic cancer vaccines

Abstract Viral vector vaccines still represent most excellent inducers of cell-mediated and humoral immune responses. Therefore, intensive investigations are performed to improve the use of several virus families as safe and efficient viral vectors, not only against diverse infectious diseases but also against tumors. During the last decade we developed a novel vector virus platform using Orf virus (ORFV), a member of the genus Parapoxvirus of Poxviridae. The attractiveness of an ORFV vector rely on the following advantages: (i) a very restricted host range, (ii) no evidence for viral systemic spread, (iii) the fast induction of humoral and cellular immune responses, especially also in non-permissive hosts that do not support vector replication, (iv) a short-term vector-specific immunity allowing multiple re-immunizations, and (v) the possibility to generate recombinants by targeted deletion of ORFV virulence genes on the basis of the highly attenuated, apathogenic ORFV strain D1701-V. Recently, we were able to demonstrate the excellent immune stimulating (humoral and cellular) and prophylactic capacity of ORFV-based recombinants against numerous different viral diseases manifold. Obviously, it would be of great interest, if ORFV might serve as a platform for the development of therapeutic tumor vaccines for humans. Hence, we would like to investigate the so far poorly understood mechanism of immune stimulation and -induction as well as the possibilities to further manipulate/trigger the induced immune response by co-expression of immune regulating factors more precise. With the aid of a recombinant ORFV expressing the fluorescent marker protein mCherry, the mode of action and the immune cells involved in this immune activation is investigated. In a first step, freshly isolated human PBMC subpopulations were infected at different time points and with different MOIs (multiplicity of infections) and the infection rate and cell viability were measured. Thereby we observed that professional antigen presenting cells (APC) were most susceptible for infection/uptake of virus. Next we wanted to investigate the activation status of the APCs. Therefore we analyzed the effect of ORFV on the expression of surface markerswhich are important for antigen-presentation and co-stimulation of T-cells (e.g. HLA DR, CD40, CD80 and CD86). In a further step we examined the cytokine release to elucidate the effects of ORFV infection on the immune stimulatory capacity of APCs. The capability to cause T-cell priming, as an integral part of the immune activation, will be studied more precisely using the model antigens Melan A and pp65 (hCMV). Further optimization of the immune stimulatory capabilities of ORFV shall be achieved by co-expression of adequate cytokines together with the integrated model antigen. Citation Format: Melanie Mueller, Ralf Amann, Thomas Feger, Hans-Georg Rammensee. The mode of action of Orf virus – a novel viral vector for therapeutic cancer vaccines. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A170.

BMET-14STAT3 INHIBITION ENHANCES THERAPEUTIC EFFICACY OF RADIATION TREATMENT AGAINST ESTABLISHED BRAIN METASTASIS IN MURINE MELANOMA MODEL

INTRODUCTION: Melanoma patients that develop brain metastasis have a median survival of 6 months. Radiation, a standard of care for local control of melanoma, has been reported to have immune stimulatory capacities. Therefore, we investigated the combinatorial effect of inhibitors of the signal transducer and activator of transcript 3 (STAT3) pathway, a potent mediator of immune suppression, and whole-brain radiation in a murine brain metastasis model. METHODS: C57BL/6 mice were intracerebrally implanted with B16 melanoma, irradiated with 7.5 Gy, and treated with an advanced lipid nanoparticle system that contained miR-124 (LNP-124) that inhibits the STAT3 pathway, or WP1066, a small molecule inhibitor of STAT3. Mice surviving 130+ days after tumor implantation were subcutaneously rechallenged with tumor. RESULTS: Monotherapy (LNP-124, WP1066, or radiation alone) demonstrated longer survival relative to untreated control mice; combination therapies further extended survival compared to single treatment agents. Median survival for mice receiving radiation combined with LNP124 or WP1066 was 65 days and 45.5 days, respectively, compared to 15 days for untreated mice and 19 - 40 days for monotherapy. While no untreated mice (control) survived over 20 days, the survival rate at day 130 for mice receiving combination therapy was almost 40%. Flow cytometry analysis showed higher IL-2 production in T cells from tumor-draining lymph nodes in mice treated with radiation plus STAT3 inhibitor. SUMMARY: These results indicate the combination of STAT3 inhibition and radiation enhances the therapeutic effect against established murine brain metastasis. The implications for this combination on antitumor immunity, immunologic memory and protection from recurrence will also be discussed.

SAMHD1 degradation enhances active suppression of dendritic cell maturation by HIV-1.

A hallmark of HIV-1 infection is the lack of sterilizing immunity. Dendritic cells (DCs) are crucial in the induction of immunity, and lack of DC activation might underlie the absence of an effective anti-HIV-1 response. We have investigated how HIV-1 infection affects maturation of DCs. Our data show that even though DCs are productively infected by HIV-1, infection does not induce DC maturation. HIV-1 infection actively suppresses DC maturation, as HIV-1 infection inhibited TLR-induced maturation of DCs and thereby decreased the immune stimulatory capacity of DCs. Interfering with SAMHD1 restriction further increased infection of DCs, but did not lead to DC maturation. Notably, higher infection observed with SAMHD1 depletion correlated with a stronger suppression of maturation. Furthermore, blocking reverse transcription rescued TLR-induced maturation. These data strongly indicate that HIV-1 replication does not trigger immune activation in DCs, but that HIV-1 escapes immune surveillance by actively suppressing DC maturation independent of SAMHD1. Elucidation of the mechanism of suppression can lead to promising targets for therapy or vaccine design.