Phenotypical Diversification of Early IFNα-Producing Human Plasmacytoid Dendritic Cells Using Droplet-Based Microfluidics

Abstract

Plasmacytoid dendritic cells (pDCs) are a rare type of highly versatile immune cells that besides their specialized function of massive type I interferon (IFN-I) production are able to exert cytotoxic effector functions. However, diversification upon toll like receptor (TLR)-induced activation leads to highly heterogeneous responses that have not been fully characterized yet. Using droplet-based microfluidics, we showed that upon TLR7/8 and TLR9-induced single-cell activation only 1-3% secretes IFNα, and only small fractions upregulate cytotoxicity markers. Interestingly, this 1-3% of early IFN-producing pDCs, also known as first responders, express high levels of programmed death-ligand 1 (PD-L1) and TNF-related apoptosis-inducing ligand (TRAIL), which makes these hybrid cells similar to earlier described IFN-I producing killer pDCs (IKpDCs). IFN-I priming increases the numbers of IFNα producing cells up to 40%, but does not significantly upregulate the cytotoxicity markers. Besides, these so-called second responders do not show a cytotoxic phenotype as potent as observed for the first responders. Overall, our results indicate that the first responders are the key drivers orchestrating population wide IFN-I responses and possess high cytotoxic potential.

Plasmacytoid dendritic cells during COVID-19: Ally or adversary?

“…This contention has also been Cell Reports 40, July 26, 2022 3 Review ll supported by studies using droplet-based microfluidic approaches, where only a limited fraction of pDCs seems to be capable of secreting IFNa. Importantly, this initial IFNa response then primes additional pDCs, via an autocrine feedback mechanism, ultimately enlarging the population of IFNa-producing pDCs (Van Eyndhoven et al, 2021;Wimmers et al, 2018). In summary, pDCs have evolved mechanisms to sense and respond to both cell-free and cell-associated viruses via a virus-sensing synapse.…”

Hydrogels for Single-Cell Microgel Production: Recent Advances and Applications

“…Droplet-based microfluidics has obtained its own prominent seat within this field due to the high-throughput and reproducible nature of the resulting microscale emulsions. An impressive resume of single-cell applications (Klein et al, 2015;Macosko et al, 2015;Wimmers et al, 2018;Bounab et al, 2020;Gérard et al, 2020;Subedi et al, 2021;van Eyndhoven et al, 2021) predicts that droplet encapsulation will continue to play an important role in isolation of individual cells over years to come. A promising addition to the field is the application of hydrogel to produce microgels for single-cell encapsulation.…”

“…Many immune responses are heavily dependent on cells being adjacent for paracrine signaling or even connecting for juxtacrine signaling. High-throughput droplet encapsulation was readily proven an ideal method to screen immune cell heterogeneity in response to paracrine ques, as modeled by co-encapsulation of various stimuli (Tiemeijer et al, 2021;Konry et al, 2011;van Eyndhoven et al, 2021;Konry et al, 2013). Co-encapsulation allows similar setups for investigating contact-dependent juxtacrine interactions referred to as immune synapses.…”

Transiently heritable fates and quorum sensing drive early IFN-I response dynamics

“…These can be manipulated by overexpression of signaling intermediates, such as retinoic acidinducible gene I (RIG-I), IRF3, and IRF7, leading to an increased overall production of IFN-Is (Harrison and Moseley, 2020;Zhao et al, 2012). In contrast, recent evidence suggests that the early phase could be dictated by determinism (Bagnall et al, 2020;Shaffer et al, 2020;Talemi and Höfer, 2018;Van Eyndhoven et al, 2021a). Accordingly, a transiently heritable gene expression program related to IFN-I signaling, including the expression of DDX58 (RIG-I), IFIT1, PMAIP1, and OASL, was discovered to be initiated only in fractions of unstimulated cells (Shaffer et al, 2020).…”

“…Over the past decades, multilayered stochasticity driving cellular heterogeneity and subsequent cellular decision-making during IFN-I responses has become increasingly apparent (Rand et al, 2012; Van Eyndhoven et al, 2021b). In short, IFN-I responses are elicited by fractions of so-called first responding cells, also referred to as ‘precocious cells’ or ‘early responding cells’, which start the initial IFN-I production upon viral detection (Bauer et al, 2016; Hjorton et al, 2020; Patil et al, 2015; Shalek et al, 2014; Van Eyndhoven et al, 2021a; Wimmers et al, 2018). Their production is further enhanced via autocrine signaling, inducing a feedforward loop resulting in the upregulation of interferon regulatory factor (IRF) 7 and other signaling components (Honda et al, 2006).…”