A cell-based microarray to investigate combinatorial effects of microparticle-encapsulated adjuvants on dendritic cell activation

Abstract

Experimental vaccine adjuvants are being designed to target specific toll-like receptors (TLRs) alone or in combination, expressed by antigen presenting cells, notably dendritic cells (DCs). There is a need for high-content screening (HCS) platforms to explore how DC activation is affected by adjuvant combinations. Presented is a cell-based microarray approach, “immunoarray”, exposing DCs to a large number of adjuvant combinations. Microparticles encapsulating TLR ligands are printed onto arrays in a range of doses for each ligand, in all possible dose combinations. Dendritic cells are then co-localized with physisorbed microparticles on the immunoarray, adherent to isolated islands surrounded by a non-fouling background, and DC activation is quantified. Delivery of individual TLR ligands was capable of eliciting high levels of specific DC activation markers. For example, either TLR9 ligand, CpG, or TLR3 ligand, poly I:C, was capable of inducing among the highest 10% expression levels of CD86. In contrast, MHC-II expression in response to TLR4 agonist MPLA was among the highest, whereas either MPLA or poly I:C, was capable of producing among the highest levels of CCR7 expression, as well as inflammatory cytokine IL-12. However, in order to produce robust responses across all activation markers, adjuvant combinations were required, and combinations were more represented among the high responders. The immunoarray also enables investigation of interactions between adjuvants, and each TLR ligand suggested antagonism to other ligands, for various markers. Altogether, this work demonstrates feasibility of the immunoarray platform to screen microparticle-encapsulated adjuvant combinations for the development of improved and personalized vaccines.

Biomaterials as Tools to Decode Immunity

“…These DCs were cultured with particles then analyzed for changes in surface protein expression or soluble cytokine secretion. Reproduced with permission . Copyright 2017, Royal Society of Chemistry.…”

“…Biomaterials can facilitate this analysis by controlling the loading concentration and subsequent codelivery of multiple encapsulated cargos. In one such example, to study dose dependent TLR interactions, microparticles (MPs) encapsulating TLR3, TLR4, and TLR9 ligands either individually or in combination were printed onto coverslips before seeding with DCs to create a cell‐based microarray (Figure g) . Using this platform, more than 200 combinations of ligands were assayed for their impact on cytokine levels and activation marker expression.…”

<strong>A General Theory of Autoimmune Disease Causation: Integrating Innate and Adaptive Immunity, Altered Antigen Processing, Sex and Genetic Predispositions, and Microbiome Effects</strong>

“…ACT proposes that complementary antigens stimulate complementary, synergistic innate mechanisms. Synergisms are well-documented within the innate immune system and include TLR2 with TLR3; TLR2 with TLR4; TLR2 with TLR6; TLR 3 with TLRs 7 and 8; TLR 4 with TLR 8 and 9 (Sato, et al, 2000;Napolitani, et al, 2005;Ghosh, et al, 2007;Krumbiegel, et al, 2007;Vanhoutte, et al, 2008;Mäkelä, et al, 2009;Lee, et al, 2016;Acharya, et al, 2016;Fischetti, et al, 2017;Nouri-Shirazi, et al, 2017). For example, TLR2 and 4 synergize in rheumatoid arthritis-derived synoviocytes Liu, et al, 2014), sarcoidosis (Wikén, et al, 2009), systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, psoriasis, multiple sclerosis, and autoimmune diabetes (Liu, et al, 2014).…”

Combinatorial drug delivery approaches for immunomodulation

“…Higher order TLR combinations are also being investigated. Recent work described high-throughput methods for combinatorial loading of factors in microparticles [56], as well as a high-throughput microarray to assess DC activation in response to combinations of three TLR-encapsulating microparticles [57,58]. The adjuvant-screening microarray demonstrated microparticle combinations delivering poly(inosinic:cytidylic acid) (poly(I:C)), MPLA, or CpG elicited differential expression of DC activation biomarkers CD86, MHC-II, CCR7, IL-12, and IL-10.…”