Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses.

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo, while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses.

CD169 Defines Activated CD14+ Monocytes With Enhanced CD8+ T Cell Activation Capacity.

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.

Mimicking Pathogens to Augment the Potency of Liposomal Cancer Vaccines.

Liposomes have emerged as interesting vehicles in cancer vaccination strategies as their composition enables the inclusion of both hydrophilic and hydrophobic antigens and adjuvants. In addition, liposomes can be decorated with targeting moieties to further resemble pathogenic particles that allow for better engagement with the immune system. However, so far liposomal cancer vaccines have not yet reached their full potential in the clinic. In this review, we summarize recent preclinical studies on liposomal cancer vaccines. We describe the basic ingredients for liposomal cancer vaccines, tumor antigens, and adjuvants, and how their combined inclusion together with targeting moieties potentially derived from pathogens can enhance vaccine immunogenicity. We discuss newly identified antigen-presenting cells in humans and mice that pose as promising targets for cancer vaccines. The lessons learned from these preclinical studies can be applied to enhance the efficacy of liposomal cancer vaccination in the clinic.

Liposomal Nanovaccine Containing α-Galactosylceramide and Ganglioside GM3 Stimulates Robust CD8+ T Cell Responses via CD169+ Macrophages and cDC1.

Successful anti-cancer vaccines aim to prime and reinvigorate cytotoxic T cells and should therefore comprise a potent antigen and adjuvant. Antigen targeting to splenic CD169+ macrophages was shown to induce robust CD8+ T cell responses via antigen transfer to cDC1. Interestingly, CD169+ macrophages can also activate type I natural killer T-cells (NKT). NKT activation via ligands such as α-galactosylceramide (αGC) serve as natural adjuvants through dendritic cell activation. Here, we incorporated ganglioside GM3 and αGC in ovalbumin (OVA) protein-containing liposomes to achieve both CD169+ targeting and superior DC activation. The systemic delivery of GM3-αGC-OVA liposomes resulted in specific uptake by splenic CD169+ macrophages, stimulated strong IFNγ production by NKT and NK cells and coincided with the maturation of cDC1 and significant IL-12 production. Strikingly, superior induction of OVA-specific CD8+ T cells was detected after immunization with GM3-αGC-OVA liposomes. CD8+ T cell activation, but not B cell activation, was dependent on CD169+ macrophages and cDC1, while activation of NKT and NK cells were partially mediated by cDC1. In summary, GM3-αGC antigen-containing liposomes are a potent vaccination platform that promotes the interaction between different immune cell populations, resulting in strong adaptive immunity and therefore emerge as a promising anti-cancer vaccination strategy.

Optimization of Liposomes for Antigen Targeting to Splenic CD169+ Macrophages.

Despite promising progress in cancer vaccination, therapeutic effectiveness is often insufficient. Cancer vaccine effectiveness could be enhanced by targeting vaccine antigens to antigen-presenting cells, thereby increasing T-cell activation. CD169-expressing splenic macrophages efficiently capture particulate antigens from the blood and transfer these antigens to dendritic cells for the activation of CD8+ T cells. In this study, we incorporated a physiological ligand for CD169, the ganglioside GM3, into liposomes to enhance liposome uptake by CD169+ macrophages. We assessed how variation in the amount of GM3, surface-attached PEG and liposomal size affected the binding to, and uptake by, CD169+ macrophages in vitro and in vivo. As a proof of concept, we prepared GM3-targeted liposomes containing a long synthetic ovalbumin peptide and tested the capacity of these liposomes to induce CD8+ and CD4+ T-cell responses compared to control liposomes or soluble peptide. The data indicate that the delivery of liposomes to splenic CD169+ macrophages can be optimized by the selection of liposomal constituents and liposomal size. Moreover, optimized GM3-mediated liposomal targeting to CD169+ macrophages induces potent immune responses and therefore presents as an interesting delivery strategy for cancer vaccination.

Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells.

Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contaminating extracellular vesicles (EV) containing RNA. Because of a high degree of sequence similarity among homologous non-coding RNAs of mammalian species, residual FCS-RNA in culture medium may interfere in the analysis of EV-RNA released by cultured cells. Recently, doubts have been raised as to whether commonly used protocols for depletion of FCS-EV efficiently remove FCS-RNA. Moreover, technical details in FCS-EV depletion protocols are known to vary between labs, which may lead to inter-study differences in contaminating FCS-RNA levels. Here, we investigated how technical modifications of EV-depletion protocols affect the efficiency with which bovine RNAs are depleted from FCS, and determined the contribution of contaminating bovine RNA to EV-RNA purified from cell cultures. Our data show differences in depletion efficiency between and within various classes of small non-coding RNA. Importantly, we demonstrate that variations in FCS-EV depletion protocols affect both the quantity and type of residual FCS-RNAs in EV-depleted medium. By using optimised FCS-EV depletion protocols combined with methods for high-grade purification of EV the levels of contaminating bovine RNA in EV populations isolated from cell culture medium can be reduced. With illustrative datasets we also demonstrate that the abundance of a specific RNA in cell culture EV can only be determined if measured relative to background levels of this RNA in medium control samples. These data highlight the need for optimisation and validation of existing and novel FCS-EV depletion methods and urge for accurate descriptions of these methods in publications to increase experimental reproducibility.