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Nano- and microparticles have increasingly widespread applications in nanomedicine, ranging from drug delivery to imaging. Poly(lactic-co-glycolic acid) (PLGA) particles are the most ...widely-applied type of particles due to their biocompatibility and biodegradability. Here, we discuss the preparation of PLGA particles, and various modifications to tailor particles for applications in biological systems. We highlight new preparation approaches, including microfluidics and PRINT method, and modifications of PLGA particles resulting in novel or responsive properties, such as Janus or upconversion particles. Finally, we describe how the preparation methods can- and should-be adapted to tailor the properties of particles for the desired biomedical application. Our aim is to enable researchers who work with PLGA particles to better appreciate the effects of the selected preparation procedure on the final properties of the particles and its biological implications.
Nanoparticles are increasingly important in the field of biomedicine. Particles made of polymers are in the spotlight, due to their biodegradability, biocompatibility, versatility. In this review, we aim to discuss the range of formulation techniques, manipulations, and applications of poly(lactic-co-glycolic acid) (PLGA) particles, to enable a researcher to effectively select or design the optimal particles for their application. We describe the various techniques of PLGA particle synthesis and their impact on possible applications. We focus on recent developments in the field of PLGA particles, and new synthesis techniques that have emerged over the past years. Overall, we show how the chemistry of PLGA particles can be adapted to solve pressing biological needs.
Toll-like receptor (TLR) agonists induce metabolic reprogramming, which is required for immune activation. We have investigated mechanisms that regulate metabolic adaptation upon TLR-stimulation in ...human blood DC subsets, CD1c
myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). We show that TLR-stimulation changes expression of genes regulating oxidative phosphorylation (OXPHOS) and glutamine metabolism in pDC. TLR-stimulation increases mitochondrial content and intracellular glutamine in an autophagy-dependent manner in pDC. TLR-induced glutaminolysis fuels OXPHOS in pDCs. Notably, inhibition of glutaminolysis and OXPHOS prevents pDC activation. Conversely, TLR-stimulation reduces mitochondrial content, OXPHOS activity and induces glycolysis in CD1c
mDC. Inhibition of mitochondrial fragmentation or promotion of mitochondrial fusion impairs TLR-stimulation induced glycolysis and activation of CD1c
mDCs. TLR-stimulation triggers BNIP3-dependent mitophagy, which regulates transcriptional activity of
α
. BNIP3-dependent mitophagy is required for induction of glycolysis and activation of CD1c
mDCs. Our findings reveal that TLR stimulation differentially regulates mitochondrial dynamics in distinct human DC subsets, which contributes to their activation.
The realization that dendritic cells (DCs) orchestrate innate and adaptive immune responses has stimulated research on harnessing DCs to create more effective vaccines. Early clinical trials ...exploring autologous DCs that were loaded with antigens ex vivo to induce T-cell responses have provided proof of principle. Here, we discuss how direct targeting of antigens to DC surface receptors in vivo might replace laborious and expensive ex vivo culturing, and facilitate large-scale application of DC-based vaccination therapies.
Immune checkpoint inhibitors propelled the field of oncology with clinical responses in many different tumor types. Superior overall survival over chemotherapy has been reported in various metastatic ...cancers. Furthermore, prolonged disease-free and overall survival have been reported in the adjuvant treatment of stage III melanoma. Unfortunately, a substantial portion of patients do not obtain a durable response. Therefore, additional strategies for the treatment of cancer are still warranted. One of the numerous options is dendritic cell vaccination, which employs the central role of dendritic cells in activating the innate and adaptive immune system. Over the years, dendritic cell vaccination was shown to be able to induce an immunologic response, to increase the number of tumor infiltrating lymphocytes and to provide overall survival benefit for at least a selection of patients in phase II studies. However, with the success of immune checkpoint inhibition in several malignancies and considering the plethora of other treatment modalities being developed, it is of utmost importance to delineate the position of dendritic cell therapy in the treatment landscape of cancer. In this review, we address some key questions regarding the integration of dendritic cell vaccination in future cancer treatment paradigms.
Dendritic cells (DCs) can initiate and direct adaptive immune responses. This ability is exploitable in DC vaccination strategies, in which DCs are educated ex vivo to present tumor antigens and are ...administered into the patient with the aim to induce a tumor-specific immune response. DC vaccination remains a promising approach with the potential to further improve cancer immunotherapy with little or no evidence of treatment-limiting toxicity. However, evidence for objective clinical antitumor activity of DC vaccination is currently limited, hampering the clinical implementation. One possible explanation for this is that the most commonly used monocyte-derived DCs may not be the best source for DC-based immunotherapy. The novel approach to use naturally circulating DCs may be an attractive alternative. In contrast to monocyte-derived DCs, naturally circulating DCs are relatively scarce but do not require extensive culture periods. Thereby, their functional capabilities are preserved, the reproducibility of clinical applications is increased, and the cells are not dysfunctional before injection. In human blood, at least three DC subsets can be distinguished, plasmacytoid DCs, CD141
and CD1c
myeloid/conventional DCs, each with distinct functional characteristics. In completed clinical trials, either CD1c
myeloid DCs or plasmacytoid DCs were administered and showed encouraging immunological and clinical outcomes. Currently, also the combination of CD1c
myeloid and plasmacytoid DCs as well as the intratumoral use of CD1c
myeloid DCs is under investigation in the clinic. Isolation and culture strategies for CD141
myeloid DCs are being developed. Here, we summarize and discuss recent clinical developments and future prospects of natural DC-based immunotherapy.
Type I interferon (IFN) is a key driver of immunity to infections and cancer. Plasmacytoid dendritic cells (pDCs) are uniquely equipped to produce large quantities of type I IFN but the mechanisms ...that control this process are poorly understood. Here we report on a droplet-based microfluidic platform to investigate type I IFN production in human pDCs at the single-cell level. We show that type I IFN but not TNFα production is limited to a small subpopulation of individually stimulated pDCs and controlled by stochastic gene regulation. Combining single-cell cytokine analysis with single-cell RNA-seq profiling reveals no evidence for a pre-existing subset of type I IFN-producing pDCs. By modulating the droplet microenvironment, we demonstrate that vigorous pDC population responses are driven by a type I IFN amplification loop. Our study highlights the significance of stochastic gene regulation and suggests strategies to dissect the characteristics of immune responses at the single-cell level.
Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, ...and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.
Dendritic cells (DCs) are central players of the immune response. To date, DC-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, predominantly with ex ...vivo-cultured monocyte-derived DCs (moDCs). However, the extensive culture period and compounds required to differentiate them into DCs may negatively affect their immunological potential. Therefore, it is attractive to consider alternative DC sources, such as blood DCs. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions. Besides scavenging pathogens and presenting antigens, DCs secrete cytokines, all of which is vital for both the acquired and the innate immune system. These immunological functions relate to Toll-like receptors (TLRs) expressed by DCs. TLRs recognize pathogen-derived products and subsequently provoke DC maturation, antigen presentation and cytokine secretion. However, not every TLR is expressed on each DC subset nor causes the same effects when activated. Considering the large amount of clinical trials using DC-based immunotherapy for cancer patients and the decisive role of TLRs in DC maturation, this review summarizes TLR expression in different DC subsets in relation to their function. Emphasis will be given to the therapeutic potential of TLR-matured DC subsets for DC-based immunotherapy.
Dendritic cells (DCs) bridge the connection between innate and adaptive immunity. DCs present antigens to T cells and stimulate potent cytotoxic T‐cell responses. Metabolic reprogramming is critical ...for DC development and activation; however, metabolic adaptations and regulation in DC subsets remains largely uncharacterized. Here, we mapped metabolomic and lipidomic signatures associated with the activation phenotype of human conventional DC type 1, a DC subset specialized in cross‐presentation and therefore of major importance for the stimulation of CD8+ T cells. Our metabolomics and lipidomic analyses showed that Toll‐like receptor (TLR) stimulation altered glycerolipids and amino acids in cDC1. Poly I:C or pRNA stimulation reduced triglycerides and cholesterol esters, as well as various amino acids. Moreover, TLR stimulation reduced expression of glycolysis‐regulating genes and did not induce glycolysis. Conversely, cDC1 exhibited increased mitochondrial content and oxidative phosphorylation (OXPHOS) upon TLR3 or TLR7/8 stimulation. Our findings highlight the metabolic adaptations required for cDC1 maturation.
Our findings highlight the metabolic adaptations required for maturation of cDC1 dendritic cells (DC). Toll like receptor stimulation alters glycerolipids and amino acids and increases mitochondrial content and oxidative phosphorylation in cDC1 DC.
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