Harnessing Dendritic Cells for Poly (D,L-lactide-co-glycolide) Microspheres (PLGA MS)—Mediated Anti-tumor Therapy

With emerging success in fighting off cancer, chronic infections, and autoimmune diseases, immunotherapy has become a promising therapeutic approach compared to conventional therapies such as surgery, chemotherapy, radiation therapy, or immunosuppressive medication. Despite the advancement of monoclonal antibody therapy against immune checkpoints, the development of safe and efficient cancer vaccine formulations still remains a pressing medical need. Anti-tumor immunotherapy requires the induction of antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses which recognize and specifically destroy tumor cells. Due to the crucial role of dendritic cells (DCs) in initiating anti-tumor immunity, targeting tumor antigens to DCs has become auspicious in modern vaccine research. Over the last two decades, micron- or nanometer-sized particulate delivery systems encapsulating tumor antigens and immunostimulatory molecules into biodegradable polymers have shown great promise for the induction of potent, specific and long-lasting anti-tumor responses in vivo. Enhanced vaccine efficiency of the polymeric micro/nanoparticles has been attributed to controlled and continuous release of encapsulated antigens, efficient targeting of antigen presenting cells (APCs) such as DCs and subsequent induction of CTL immunity. Poly (D, L-lactide-co-glycolide) (PLGA), as one of these polymers, has been extensively studied for the design and development of particulate antigen delivery systems in cancer therapy. This review provides an overview of the current state of research on the application of PLGA microspheres (PLGA MS) as anti-tumor cancer vaccines in activating and potentiating immune responses attempting to highlight their potential in the development of cancer therapeutics

Immunoproteasome Inhibition Impairs T and B Cell Activation by Restraining ERK Signaling and Proteostasis

Immunoproteasome (IP) inhibition holds potential as a novel treatment option for various immune-mediated pathologies. The IP inhibitor ONX 0914 reduced T cell cytokine secretion and Th17 polarization and showed pre-clinical efficacy in a range of autoimmune disorders, transplant-allograft rejection, virus-mediated tissue damage, and colon cancer progression. However, the molecular basis of these effects has remained largely elusive. Here, we have analyzed the effects of ONX 0914 in primary human and mouse lymphocytes. ONX 0914-treatment impaired primary T cell activation in vitro and in vivo. IP inhibition reduced ERK-phosphorylation sustainment, while leaving NF-κB and other signaling pathways unaffected. Naïve T and B cells expressed nearly exclusively immuno- or mixed proteasomes but no standard proteasomes and IP inhibition but not IP-deficiency induced mild proteostasis stress, reduced DUSP5 expression and enhanced DUSP6 protein levels due to impaired degradation. However, accumulation of DUSP6 did not cause the reduced ERK-phosphorylation in a non-redundant manner. We show that broad-spectrum proteasome inhibition and immunoproteasome inhibition have distinct effects on T cell activation at the molecular level. Notably, ONX 0914-treated T cells recovered from proteostasis stress without apoptosis induction, apparently via Nrf1-mediated up-regulation of standard proteasomes. In contrast, B cells were more susceptible to apoptosis after ONX 0914-treatment. Our data thus provide mechanistic insights how IP inhibition functionally impedes T and B cells likely accounting for its therapeutic benefits

A Single Residue Exchange Within a Viral CTL Epitope Alters Proteasome-Mediated Degradation Resulting in Lack of Antigen Presentation

AbstractCTL epitope (KSPWFTTL) encoded by AKV/MCF type of murine leukemia virus (MuLV) differs from the sequence in Friend/Moloney/Rauscher (FMR) type in one residue (RSPWFTTL). CTL experiments indicated defective processing of the FMR peptide in tumor cells. Proteasome-mediated digestion of AKV/MCF-type 26-mer peptides resulted in the early generation and higher levels of epitope-containing fragments than digestion of FMR-type peptides, explained by prominent cleavage next to R in the FMR sequence. The fragments were identified as 10- and 11-mer peptides and were efficiently translocated by TAP. The naturally presented AKV/MCF peptide is the 8-mer, indicating ER peptide trimming. In conclusion, a single residue exchange can cause CTL epitope destruction by specific proteasomal cleavage

Novel intranasal vaccine targeting SARS-CoV-2 receptor binding domain to mucosal microfold cells and adjuvanted with TLR3 agonist Riboxxim™ elicits strong antibody and T-cell responses in mice

SARS-CoV-2 continues to circulate in the human population necessitating regular booster immunization for its long-term control. Ideally, vaccines should ideally not only protect against symptomatic disease, but also prevent transmission via asymptomatic shedding and cover existing and future variants of the virus. This may ultimately only be possible through induction of potent and long-lasting immune responses in the nasopharyngeal tract, the initial entry site of SARS-CoV-2. To this end, we have designed a vaccine based on recombinantly expressed receptor binding domain (RBD) of SARS-CoV-2, fused to the C-terminus of C. perfringens enterotoxin, which is known to target Claudin-4, a matrix molecule highly expressed on mucosal microfold (M) cells of the nasal and bronchial-associated lymphoid tissues. To further enhance immune responses, the vaccine was adjuvanted with a novel toll-like receptor 3/RIG-I agonist (Riboxxim™), consisting of synthetic short double stranded RNA. Intranasal prime-boost immunization of mice induced robust mucosal and systemic anti-SARS-CoV-2 neutralizing antibody responses against SARS-CoV-2 strains Wuhan-Hu-1, and several variants (B.1.351/beta, B.1.1.7/alpha, B.1.617.2/delta), as well as systemic T-cell responses. A combination vaccine with M-cell targeted recombinant HA1 from an H1N1 G4 influenza strain also induced mucosal and systemic antibodies against influenza. Taken together, the data show that development of an intranasal SARS-CoV-2 vaccine based on recombinant RBD adjuvanted with a TLR3 agonist is feasible, also as a combination vaccine against influenza

The inherited blindness protein AIPL1 regulates the ubiquitin-like FAT10 pathway

Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway

No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

The proteasome is the main protein-degrading machine within the cell, producing ligands for MHC class I molecules. It is a cylindrical multicatalytic protease complex, and the catalytic activity is mediated by the three subunits β1, β2, and β5 which possess caspase-, trypsin-, and chymotrypsin-like activities, respectively. By stimulation with interferon (IFN)-γ the replacement of these subunits by β1i, β2i, and β5i is induced leading to formation of immunoproteasomes with altered proteolytic and antigen processing properties. The genes coding for these immunosubunits are restricted to jawed vertebrates but have so far not been found in the genomes of birds, e.g., chicken, turkey, quail, black grouse and zebra finch. However, the chicken genome sequences are not completely assigned; therefore, we investigated the presence of immunoproteasome on protein level. 20S proteasome was purified from the chicken brain, blood, spleen, and bursa of Fabricius, followed by separation via two-dimensional (2D) gel electrophoresis. We analyzed the protein spots derived from the spleen and brain by mass spectrometry and could identify all 14 proteasomal subunits, but there were no differences detectable in the spot patterns. Moreover, we stimulated the chicken spleen cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin aiming at the induction of immunoproteasome, but in spite of the induction of proliferation and IFN-γ, no evidence for immunoproteasome formation in chicken could be obtained. This result was substantiated by the finding that 20S proteasomes isolated from immune and non-immune tissues showed very similar peptidolytic activities. Taken together, our results indicate that chicken lack immunoproteasomes also on protein level

The ubiquitin-like modifier FAT10 : much more than a proteasome-targeting signal

Human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10) also called ubiquitin D (UBD) is a member of the ubiquitin-like modifier (ULM) family. The FAT10 gene is localized in the MHC class I locus and FAT10 protein expression is mainly restricted to cells and organs of the immune system. In all other cell types and tissues, FAT10 expression is highly inducible by the pro-inflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF). Besides ubiquitin, FAT10 is the only ULM which directly targets its substrates for degradation by the 26S proteasome. This poses the question as to why two ULMs sharing the proteasome-targeting function have evolved and how they differ from each other. This Review summarizes the current knowledge of the special structure of FAT10 and highlights its differences from ubiquitin. We discuss how these differences might result in differential outcomes concerning proteasomal degradation mechanisms and non-covalent target interactions. Moreover, recent insights about the structural and functional impact of FAT10 interacting with specific non-covalent interaction partners are reviewed.publishe