Chemical synthesis and immunological evaluation of cancer vaccines based on ganglioside antigens and α-galactosylceramide.

iNKT cells - often referred as the "Swiss Army knife" of the immune system - have emerged as central players in cancer vaccine therapies. Glycolipids activating iNKT cells, such as α-galactosylceramide (αGalCer), can enhance the immune response against co-delivered cancer antigens and have been applied in the design of self-adjuvanting anti-tumor vaccines. In this context, this work focuses on the chemical synthesis of ganglioside tumor-associated carbohydrate antigens (TACAs), namely GM3 and (Neu5Gc)GM3 antigens, their conjugation to αGalCer, and their formulation into liposomes as an efficient platform for their in vivo delivery. Liposomes containing GM3-αGalCer, (Neu5Gc)GM3-αGalCer, and equimolar amounts of the two conjugates have been fully characterized and their ability to activate iNKT cell has been confirmed ex vivo in mouse and human cell assays. The candidates were tested in in vivo immunization studies, demonstrating an ability to induce both TH1 and TH2 cytokines leading to the production of all subclasses of IgG antibodies. Notably, the study also demonstrated that serum antibodies raised against the two TACAs, alone and in combination, were cross-reactive. This finding has consequences for future vaccine designs - even if a highly tumor-selective antigen is chosen, the resulting antibody response may be broader than anticipated.

Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

Clinical Staphylococcus aureus inhibits human T-cell activity through interaction with the PD-1 receptor.

IMPORTANCE: Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus (S. aureus) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus-imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections.

Combining Elements from Two Antagonists of Formyl Peptide Receptor 2 Generates More Potent Peptidomimetic Antagonists.

Structural optimization of a peptidomimetic antagonist of formyl peptide receptor 2 (FPR2) was explored by an approach involving combination of elements from the two most potent FPR2 antagonists described: a Rhodamine B-conjugated 10-residue gelsonin-derived peptide (i.e., PBP10, RhB-QRLFQVKGRR-OH) and the palmitoylated α-peptide/ß-peptoid hybrid Pam-(Lys-ßNspe)6-NH2. This generated an array of hybrid compounds from which a new subclass of receptor-selective antagonists was identified. The most potent representatives displayed activity in the low nanomolar range. The resulting stable and potent FPR2-selective antagonists (i.e., RhB-(Lys-ßNphe)n-NH2; n = 4-6) are expected to become valuable tools in further elucidation of the physiological role of FPR2 in health and disease.

Anti-inflammatory Properties of Antimicrobial Peptides and Peptidomimetics: LPS and LTA Neutralization.

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) neutralization constitute potential non-antibiotic treatment strategies for sepsis - a systemic infection-induced inflammatory response. Studies on LPS- and LTA-neutralizing compounds are abundant in literature, and a number of peptides and peptidomimetics appear to display promising activity. However, in this ongoing search for potential antisepsis drug leads, it will be preferable that the assays used by different research groups lead to readily comparable data for the most efficient compounds. Here, we propose and describe standardized methods to be used for testing of novel compounds for their LPS- and LTA-neutralizing capacity with a focus on functional suppression of pro-inflammatory responses in cell-based systems. To best mimic the human in vivo conditions, we suggest the use of freshly isolated human leukocytes combined with an appropriate method for the chosen cytokine (e.g., IL-6 or TNF-α). The described protocols comprise isolation, stimulation, and viability test of the human leukocytes.

N-glycosylation of asparagine 8 regulates surface expression of major histocompatibility complex class I chain-related protein A (MICA) alleles dependent on threonine 24.

NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn(8)) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr(24)) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions.

2-deoxy D-glucose prevents cell surface expression of NKG2D ligands through inhibition of N-linked glycosylation.

NKG2D ligand surface expression is important for immune recognition of stressed and neotransformed cells. In this study, we show that surface expression of MICA/B and other NKG2D ligands is dependent on N-linked glycosylation. The inhibitor of glycolysis and N-linked glycosylation, 2-deoxy-D-glucose (2DG), potently inhibited surface expression of MICA/B after histone deacetylase inhibitor treatment; the inhibition occurred posttranscriptionally without affecting MICA promoter activity. Transient overexpression of MICA surface expression was also inhibited by 2DG. 2DG blocks N-linked glycosylation of MICA/B by a reversible mechanism that can be alleviated by addition of d-mannose; this does not, however, affect the inhibition of glycolysis. Addition of d-mannose restored MICA/B surface expression after 2DG treatment. In addition, specific pharmacological or small interfering RNA-mediated targeting of glycolytic enzymes did not affect MICA/B surface expression, strongly suggesting that N-linked glycosylation, and not glycolysis, is essential for MICA/B surface expression. Corroborating this, tunicamycin, a selective inhibitor of N-linked glycosylation, abolished MICA/B surface expression without compromising activation of MICA promoter activity. NK cell-mediated killing assay and staining with a recombinant NKG2D-Fc fusion protein showed that all functional NKG2D ligands induced by histone deacetylase inhibitor treatment were abolished by 2DG treatment and fully reconstituted by further addition of d-mannose. Our data suggest that posttranslational N-linked glycosylation is strictly required for NKG2D ligand surface expression. Cancer and infection often result in aberrant glycosylation, which could likely be involved in modulation of NKG2D ligand expression. Our data further imply that chemotherapeutic use of 2DG may restrict NKG2D ligand surface expression and inhibit secretion of immunoinhibitory soluble NKG2D ligands.