Both Type I and Type II Interferons Can Activate Antitumor M1 Macrophages When Combined With TLR Stimulation

Triggering or enhancing antitumor activity of tumor-associated macrophages is an attractive strategy for cancer treatment. We have previously shown that the cytokine interferon-γ (IFN-γ), a type II IFN, could synergize with toll-like receptor (TLR) agonists for induction of antitumor M1 macrophages. However, the toxicity of IFN-γ limits its clinical use. Here, we investigated whether the less toxic type I IFNs, IFN-α, and IFN-β, could potentially replace IFN-γ for induction of antitumor M1 macrophages. We measured in vitro the ability of type I and II IFNs to synergize with TLR agonists for transcription of inducible nitric oxide synthase (iNOS) mRNA and secretion of nitric oxide (NO) by mouse bone marrow-derived macrophages (BMDMs). An in vitro growth inhibition assay was used to measure both cytotoxic and cytostatic activity of activated macrophages against Lewis lung carcinoma (LLC) cancer cells. We found that both type I and II IFNs could synergize with TLR agonists in inducing macrophage-mediated inhibition of cancer cell growth, which was dependent on NO. The ability of high dose lipopolysaccharide (LPS) to induce tumoricidal activity in macrophages in the absence of IFN-γ was shown to depend on induction of autocrine type I IFNs. Antitumor M1 macrophages could also be generated in the absence of IFN-γ by a combination of two TLR ligands when using the TLR3 agonist poly(I:C) which induces autocrine type I IFNs. Finally, we show that encapsulation of poly(I:C) into nanoparticles improved its potency to induce M1 macrophages up to 100-fold. This study reveals the potential of type I IFNs for activation of antitumor macrophages and indicates new avenues for cancer immunotherapy based on type I IFN signaling, including combination of TLR agonists

A human endothelial cell-based recycling assay for screening of FcRn targeted molecules.

Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening. In HERA, rescue from degradation depends on FcRn, and engineered ligands are recycled in a manner that correlates with their half-lives in human FcRn transgenic mice. Thus, HERA is a novel cellular assay that can be used to predict how FcRn-binding proteins are rescued from intracellular degradation. Nat Commun 2018 Feb 12; 9(1):621

Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells

Background Chemokines are attractive candidates for vaccine adjuvants due to their ability to recruit the immune cells. Lactic acid bacteria (LAB)-based delivery vehicles have potential to be used as a cheap and safe option for vaccination. Chemokine produced on the surface of LAB may potentially enhance the immune response to an antigen and this approach can be considered in development of future mucosal vaccines. Results We have constructed strains of Lactobacillus plantarum displaying a chemokine on their surface. L. plantarum was genetically engineered to express and anchor to the surface a protein called CCL3Gag. CCL3Gag is a fusion protein comprising of truncated HIV-1 Gag antigen and the murine chemokine CCL3, also known as MIP-1α. Various surface anchoring strategies were explored: (1) a lipobox-based covalent membrane anchor, (2) sortase-mediated covalent cell wall anchoring, (3) LysM-based non-covalent cell wall anchoring, and (4) an N-terminal signal peptide-based transmembrane anchor. Protein production and correct localization were confirmed using Western blotting, flow cytometry and immunofluorescence microscopy. Using a chemotaxis assay, we demonstrated that CCL3Gag-producing L. plantarum strains are able to recruit immune cells in vitro. Conclusions The results show the ability of engineered L. plantarum to produce a functional chemotactic protein immobilized on the bacterial surface. We observed that the activity of surface-displayed CCL3Gag differed depending on the type of anchor used. The chemokine which is a part of the bacteria-based vaccine may increase the recruitment of immune cells and, thereby, enhance the reaction of the immune system to the vaccine

Accounting Problems Under the Excess Profits Tax

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV- 1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8(+) T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.Funding Agencies|Research Council of Norway; Odd Fellow</p

Immune Cell Composition in Human Non-small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the world. Immunological analysis of the tumor microenvironment (immunoscore) shows great promise for improved prognosis and prediction of response to immunotherapy. However, the exact immune cell composition in NSCLC remains unclear. Here, we used flow cytometry to characterize the immune infiltrate in NSCLC tumors, non-cancerous lung tissue, regional lymph node, and blood. The cellular identity of &gt;95% of all CD45+ immune cells was determined. Thirteen distinct immune cell types were identified in NSCLC tumors. T cells dominated the lung cancer landscape (on average 47% of all CD45+ immune cells). CD4+ T cells were the most abundant T cell population (26%), closely followed by CD8+ T cells (22%). Double negative CD4−CD8− T cells represented a small fraction (1.4%). CD19+ B cells were the second most common immune cell type in NSCLC tumors (16%), and four different B cell sub-populations were identified. Macrophages and natural killer (NK) cells composed 4.7 and 4.5% of the immune cell infiltrate, respectively. Three types of dendritic cells (DCs) were identified (plasmacytoid DCs, CD1c+ DCs, and CD141+ DCs) which together represented 2.1% of all immune cells. Among granulocytes, neutrophils were frequent (8.6%) with a high patient-to-patient variability, while mast cells (1.4%), basophils (0.4%), and eosinophils (0.3%) were less common. Across the cohort of patients, only B cells showed a significantly higher representation in NSCLC tumors compared to the distal lung. In contrast, the percentages of macrophages and NK cells were lower in tumors than in non-cancerous lung tissue. Furthermore, the fraction of macrophages with high HLA-DR expression levels was higher in NSCLC tumors relative to distal lung tissue. To make the method readily accessible, antibody panels and flow cytometry gating strategy used to identify the various immune cells are described in detail. This work should represent a useful resource for the immunomonitoring of patients with NSCLC

DNA vaccines: MHC II-targeted vaccine protein produced by transfected muscle fibres induces a local inflammatory cell infiltrate in mice

Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45+, MHC class II+ and CD11b+ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry+ muscle fibers was dependent on the MHC II-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity

Oligomerized, filamentous surface presentation of RANTES/CCL5 on vascular endothelial cells

Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins. It appears that several chemokines must form higher-order oligomers to elicit proper in vivo effects, as mutants restricted to forming dimers have lost the ability to recruit leukocytes to sites of inflammation. Here, we show for the first time that the chemokine RANTES/CCL5 binds to the surface of human endothelial cells in a regular filamentous pattern. Furthermore, the filaments bound to the surface in a heparan sulfate-dependent manner. By electron microscopy we observed labeling for RANTES on membrane projections as well as on the remaining plasma membrane. Mutant constructs of RANTES restricted either in binding to heparin, or in forming dimers or tetramers, appeared either in a granular, non-filamentous pattern or were not detectable on the cell surface. The RANTES filaments were also present after exposure to flow, suggesting that they can be present in vivo. Taken together with the lacking in vivo or in vitro effects of RANTES mutants, we suggest that the filamentous structures of RANTES may be of physiological importance in leukocyte recruitment

Antibodies utilized in ELISA, Western blotting, and immunostaining.

<p>m, mouse; h, human; AP, alkaline phosphatase.</p