Preclinical evaluation of therapeutic vaccines for chronic hepatitis B that stimulate antiviral activities of T cells and NKT cells.

Background & Aims: Liver diseases resulting from chronic HBV infection are a significant cause of morbidity and mortality. Vaccines that elicit T-cell responses capable of controlling the virus represent a treatment strategy with potential for long-term effects. Here, we evaluated vaccines that induce the activity of type I natural killer T (NKT) cells to limit viral replication and license stimulation of conventional antiviral T-cells.

Tumor-targeted therapy with BRAF-inhibitor recruits activated dendritic cells to promote tumor immunity in melanoma.

Background: Tumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially dendritic cells (DC) during tumor-targeted therapy.

Methods: Here, we investigated therapy-mediated immunological alterations in the tumor microenvironment (TME) and tumor-draining lymph nodes (LN) in the D4M.

mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria.

Synthesis and Biological Evaluation of Peptide-Adjuvant Conjugate Vaccines with Increasing Antigen Content.

Synthetic vaccines that induce T cell responses to peptide epitopes are a promising immunotherapy for both communicable and noncommunicable diseases. Stimulating strong and sustained T cell responses requires antigen delivery to appropriately activated antigen presenting cells (APCs). One way this can be accomplished is by chemically conjugating immunogenic peptide epitopes with α-galactosylceramide (α-GalCer), a glycolipid that acts as an immune adjuvant by inducing stimulatory interactions between APCs and type I natural killer T (NKT) cells.

MAIT cells activate dendritic cells to promote T cell differentiation and induce humoral immunity.

Protective immune responses against respiratory pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, are initiated by the mucosal immune system. However, most licensed vaccines are administered parenterally and are largely ineffective at inducing mucosal immunity. The development of safe and effective mucosal vaccines has been hampered by the lack of a suitable mucosal adjuvant.

Using Full-Spectrum Flow Cytometry to Phenotype Memory T and NKT Cell Subsets with Optimized Tissue-Specific Preparation Protocols.

Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ.

Glycolipid-peptide vaccination induces liver-resident memory CD8 T cells that protect against rodent malaria.

Liver resident-memory CD8 T cells (T cells) can kill liver-stage -infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver T cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate -specific CD8 T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer).

The Synthesis and Anti-tumour Properties of Poly Ethoxy Ethyl Glycinamide (PEE-G) Scaffolds with Multiple PD-1 Peptides Attached.

Multivalent structures can provide multiple interactions at a target site and improve binding affinity. The multivalent presentation of the anti-tumour heptapeptide, SNTSESF, was investigated. This peptide's activity has been attributed to blockade of the PD-1 receptor-mediated signalling pathway.

Augmenting Influenza-Specific T Cell Memory Generation with a Natural Killer T Cell-Dependent Glycolipid-Peptide Vaccine.

The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective.

Activated NKT Cells Can Condition Different Splenic Dendritic Cell Subsets To Respond More Effectively to TLR Engagement and Enhance Cross-Priming.

The function of dendritic cells (DCs) can be modulated through multiple signals, including recognition of pathogen-associated molecular patterns, as well as signals provided by rapidly activated leukocytes in the local environment, such as innate-like T cells. In this article, we addressed the possibility that the roles of different murine DC subsets in cross-priming CD8(+) T cells can change with the nature and timing of activatory stimuli. We show that CD8α(+) DCs play a critical role in cross-priming CD8(+) T cell responses to circulating proteins that enter the spleen in close temporal association with ligands for TLRs and/or compounds that activate NKT cells.

Enhanced immunosuppression by therapy-exposed glioblastoma multiforme tumor cells.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an extremely short time to relapse following standard treatment. Since recurrent GBM is often resistant to subsequent radiotherapy and chemotherapy, immunotherapy has been proposed as an alternative treatment option. Although it is well established that GBM induces immune suppression, it is currently unclear what impact prior conventional therapy has on the ability of GBM cells to modulate the immune environment.

Vaccination with irradiated tumor cells pulsed with an adjuvant that stimulates NKT cells is an effective treatment for glioma.

Purpose: The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor despite recent treatment advances. There is an urgent need to develop novel therapies for this disease.

Experimental Design: We used the implantable GL261 murine glioma model to investigate the therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed with the immuno-adjuvant α-galactosylceramide (α-GalCer).

Immature murine NKT cells pass through a stage of developmentally programmed innate IL-4 secretion.

We assessed the production of the canonical Th2 cytokine IL-4 by NKT cells directly in vivo using IL-4-substituting strains of reporter mice that provide faithful and sensitive readouts of cytokine production without the confounding effects of in vitro stimulation. Analysis in naïve animals revealed an "innate" phase of IL-4 secretion that did not need to be triggered by administration of a known NKT cell ligand. This secretion was by immature NKT cells spanning Stage 1 of the maturation process in the thymus (CD4(+) CD44(lo) NK1.

Side population is not necessary or sufficient for a cancer stem cell phenotype in glioblastoma multiforme.

There is strong evidence for the existence of cancer stem cells (CSCs) in the aggressive brain tumor glioblastoma multiforme (GBM). These cells have stem-like self-renewal activity and increased tumor initiation capacity and are believed to be responsible for recurrence due to their resistance to therapy. Several techniques have been used to enrich for CSC, including growth in serum-free defined media to induce sphere formation, and isolation of a stem-like cell using exclusion of the fluorescent dye Hoechst 33342, the side population (SP).

Administration of alpha-galactosylceramide impairs the survival of dendritic cell subpopulations in vivo.

In this study, we examine whether recognition of α-GalCer presented on CD1d-expressing DCs and B cells in vivo elicits the cytotoxic activity of iNKT cells and elimination of α-GalCer-presenting cells. We report that i.v.

Potent anti-tumor responses to immunization with dendritic cells loaded with tumor tissue and an NKT cell ligand.

Cancer immunotherapy is well tolerated and specific, but its efficacy remains variable. To enhance anti-tumor CD8(+) T-cell responses induced by immunization with antigen-loaded dendritic cells (DCs), we explored the impact of eliciting a potent source of T-cell help from activated invariant natural killer (NK)-like T cells (iNKT cells) using the specific glycolipid ligand alpha-galactosylceramide (alpha-GalCer). As cytokines released by iNKT cells may drive proliferation of CD4(+)CD25(+) regulatory T cells (Tregs), we assessed this immunization strategy in animals treated with anti-CD25 antibody to inactivate Treg function.

Langerin+ CD8alpha+ dendritic cells are critical for cross-priming and IL-12 production in response to systemic antigens.

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8alpha(+) DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8(+) T cells, while CD8alpha(-) DC are more likely to present extracellular Ags on MHC class II molecules to CD4(+) T cells. As a proportion of CD8alpha(+) DC have been shown to express langerin (CD207), we investigated the role of langerin(+)CD8alpha(+) DC in presenting Ag and priming T cell responses to soluble Ags.

Targeting antigen to MHC class II molecules promotes efficient cross-presentation and enhances immunotherapy.

An efficient pathway of cross-presentation common to a range of dendritic cell (DC) populations was identified by targeting Ag to MHC class II molecules. This finding was achieved by conjugating Ag to M1, which is a modified version of the superantigen streptococcal mitogenic exotoxin Z-2 that binds to MHC class II molecules but cannot directly stimulate T cells. M1 conjugates were efficiently presented to CD4(+) and CD8(+) T cells by bone marrow-derived DC and Langerhans cells in vitro.

Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells.

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide.

Novel synthesis of alpha-galactosyl-ceramides and confirmation of their powerful NKT cell agonist activity.

alpha-Galactosyl-ceramide (1) has been identified as a powerful modulator of immunological processes through its capacity to bind CD1d molecules and specifically activate invariant natural killer (NK)-like T cells (iNKT cells). This paper describes the synthesis of 1, the analogous alpha-galactosyl-ceramide 3, and its short chain analogue 'OCH' (2), by use of the 4,6-di-O-tert-butylsilylene (DTBS) protecting group to produce a powerful alpha-galactosylating agent. In vivo experiments confirmed these compounds to be potent and selective activators of iNKT cells in a CD1d-dependent manner, each inducing a unique profile of cytokine release.

Dendritic cells loaded with stressed tumor cells elicit long-lasting protective tumor immunity in mice depleted of CD4+CD25+ regulatory T cells.

Dendritic cell (DC)-based vaccination represents a promising approach to harness the specificity and potency of the immune system to combat cancer. Finding optimal strategies for tumor Ag preparation and subsequent pulsing of DC, as well as improving the immunogenicity of weak tumor Ags remain among the first challenges of this approach. In this report, we use a prophylactic vaccine consisting of DC loaded with whole, nonmanipulated B16-F10 melanoma cells that had been stressed by heat shock and gamma irradiation.

High resolution loss of heterozygosity mapping of 17p13 in thyroid cancer: Hurthle cell carcinomas exhibit a small 411-kilobase common region of allelic imbalance, probably containing a novel tumor suppressor gene.

There is strong evidence in many tumor types, including thyroid cancer, for a novel tumor suppressor gene (TSG) at 17p13. To identify the putative thyroid 17p13 TSG we mapped thyroid tumor loss of heterozygosity (LOH) at high resolution within this region. We examined 20 typical follicular thyroid carcinomas (FTC), 19 Hurthle cell carcinomas (HCC), 15 papillary thyroid carcinomas (PTC), and 7 follicular adenomas (FA) for LOH at 17p13 using 18 probes.

Loss of heterozygosity studies revisited: prior quantification of the amplifiable DNA content of archival samples improves efficiency and reliability.

Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) studies of archival formalin-fixed, paraffin-embedded (FFPE) tumor tissues have become an important tool in the search for tumor suppressor genes and oncogenes and are also used increasingly in clinical practice. However, FFPE tissue samples may contain little amplifiable DNA, resulting in frequent reaction failures and unreliable LOH data. Using pairs of serial dilutions of reference DNA, we determined the minimum amplifiable DNA concentration necessary for reliable microsatellite-PCR LOH analysis.