Immune quorum sensing dictates IFN-I response dynamics in human plasmacytoid dendritic cells.

Type I interferons (IFN-Is) are key in fighting viral infections, but also serve major roles beyond antiviral immunity. Crucial is the tight regulation of IFN-I responses, while excessive levels are harmful to the cells. In essence, immune responses are generated by single cells making their own decisions, which are based on the signals they perceive. Additionally, immune cells must anticipate the future state of their environment, thereby weighing the costs and benefits of each possible outcome, in the presence of other potentially competitive decision makers (i.e., IFN-I producing cells). A rather new cellular communication mechanism called quorum sensing describes the effect of cell density on cellular secretory behaviors, which fits well with matching the right amount of IFN-Is produced to fight an infection. More competitive decision makers must contribute relatively less and vice versa. Intrigued by this concept, we assessed the effects of immune quorum sensing in pDCs, specialized immune cells known for their ability to mass produce IFN-Is. Using conventional microwell assays and droplet-based microfluidics assays, we were able the characterize the effect of quorum sensing in human primary immune cells in vitro. These insights open new avenues to manipulate IFN-I response dynamics in pathological conditions affected by aberrant IFN-I signaling.

Unraveling IFN-I response dynamics and TNF crosstalk in the pathophysiology of systemic lupus erythematosus.

Introduction: The innate immune system serves the crucial first line of defense against a wide variety of potential threats, during which the production of pro-inflammatory cytokines IFN-I and TNFα are key. This astonishing power to fight invaders, however, comes at the cost of risking IFN-I-related pathologies, such as observed during autoimmune diseases, during which IFN-I and TNFα response dynamics are dysregulated. Therefore, these response dynamics must be tightly regulated, and precisely matched with the potential threat. This regulation is currently far from understood. Methods: Using droplet-based microfluidics and ODE modeling, we studied the fundamentals of single-cell decision-making upon TLR signaling in human primary immune cells (n = 23). Next, using biologicals used for treating autoimmune diseases [i.e., anti-TNFα, and JAK inhibitors], we unraveled the crosstalk between IFN-I and TNFα signaling dynamics. Finally, we studied primary immune cells isolated from SLE patients (n = 8) to provide insights into SLE pathophysiology. Results: single-cell IFN-I and TNFα response dynamics display remarkable differences, yet both being highly heterogeneous. Blocking TNFα signaling increases the percentage of IFN-I-producing cells, while blocking IFN-I signaling decreases the percentage of TNFα-producing cells. Single-cell decision-making in SLE patients is dysregulated, pointing towards a dysregulated crosstalk between IFN-I and TNFα response dynamics. Discussion: We provide a solid droplet-based microfluidic platform to study inherent immune secretory behaviors, substantiated by ODE modeling, which can challenge the conceptualization within and between different immune signaling systems. These insights will build towards an improved fundamental understanding on single-cell decision-making in health and disease.

Engineering global and local signal generators for probing temporal and spatial cellular signaling dynamics.

Cells constantly encounter a wide range of environmental signals and rely on their signaling pathways to initiate reliable responses. Understanding the underlying signaling mechanisms and cellular behaviors requires signal generators capable of providing diverse input signals to deliver to cell systems. Current research efforts are primarily focused on exploring cellular responses to global or local signals, which enable us to understand cellular signaling and behavior in distinct dimensions. This review presents recent advancements in global and local signal generators, highlighting their applications in studying temporal and spatial signaling activity. Global signals can be generated using microfluidic or photochemical approaches. Local signal sources can be created using living or artificial cells in combination with different control methods. We also address the strengths and limitations of each signal generator type, discussing challenges and potential extensions for future research. These approaches are expected to continue to facilitate on-going research to discover novel and intriguing cellular signaling mechanisms.

Single-cell analysis reveals TLR-induced macrophage heterogeneity and quorum sensing dictate population wide anti-inflammatory feedback in response to LPS.

The role of macrophages in controlling tissue inflammation is indispensable to ensure a context-appropriate response to pathogens whilst preventing excessive tissue damage. Their initial response is largely characterized by high production of tumor necrosis factor alpha (TNFα) which primes and attracts other immune cells, thereafter, followed by production of interleukin 10 (IL-10) which inhibits cell activation and steers towards resolving of inflammation. This delicate balance is understood at a population level but how it is initiated at a single-cell level remains elusive. Here, we utilize our previously developed droplet approach to probe single-cell macrophage activation in response to toll-like receptor 4 (TLR4) stimulation, and how single-cell heterogeneity and cellular communication affect macrophage-mediated inflammatory homeostasis. We show that only a fraction of macrophages can produce IL-10 in addition to TNFα upon LPS-induced activation, and that these cells are not phenotypically different from IL-10 non-producers nor exhibit a distinct transcriptional pathway. Finally, we demonstrate that the dynamics of TNFα and IL-10 are heavily controlled by macrophage density as evidenced by 3D hydrogel cultures suggesting a potential role for quorum sensing. These exploratory results emphasize the relevance of understanding the complex communication between macrophages and other immune cells and how these amount to population-wide responses.

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

Type I interferon (IFN-I)-mediated antiviral responses are central to host defense against viral infections. Crucial is the tight and well-orchestrated control of cellular decision-making leading to the production of IFN-Is. Innovative single-cell approaches revealed that the initiation of IFN-I production is limited to only fractions of 1-3% of the total population, both found in vitro, in vivo, and across cell types, which were thought to be stochastically regulated. To challenge this dogma, we addressed the influence of various stochastic and deterministic host-intrinsic factors on dictating early IFN-I responses, using a murine fibroblast reporter model. Epigenetic drugs influenced the percentage of responding cells. Next, with the classical Luria-Delbrück fluctuation test, we provided evidence for transient heritability driving responder fates, which was verified with mathematical modeling. Finally, while studying varying cell densities, we substantiated an important role for cell density in dictating responsiveness, similar to the phenomenon of quorum sensing. Together, this systems immunology approach opens up new avenues to progress the fundamental understanding on cellular decision-making during early IFN-I responses, which can be translated to other (immune) signaling systems.

When we start to develop a cold, influenza or another viral infection, some of our cells produce signaling molecules known as type I interferons (or IFN-Is for short). These early IFN-I signals establish defenses against viruses in both infected and as yet uninfected cells. If the cells produce too much IFN-Is, however, it can result in uncontrolled inflammation that may harm the body and cause life threatening illness. Individual cells need to tightly control how much IFN-Is they produce and match this with the course of the viral infection. They also need to assess how much IFN-I their neighbors are producing and adjust their behavior accordingly. Cells have evolved a myriad of mechanisms to ensure the right amounts of IFN-Is are produced in different circumstances. Broadly, these mechanisms can be divided into two categories: stochastic regulation and deterministic regulation. Stochastic regulation occurs when individual cells receive the exact same information, but this leads to different outcomes, such as, different cells producing various quantities of IFN-Is. In contrast, deterministic regulation causes the same outcome in different cells independent on the information they receive. It was thought that stochastic regulation is the main driver of early IFN-1 responses, but recently a handful of studies have reported deterministic regulation being primarily responsible, instead. Here, Van Eyndhoven et al. explored the roles of both types of regulation in the early IFN-I responses of mouse cells. Van Eyndhoven et al. used genetic approaches and mathematical modelling to show that the fraction of cells that initiate early IFN-I responses can be considered deterministic. Moreover, this deterministic feature turned out to be heritable, such that the fate to produce IFN-I gets passed on for several generations of cells. Additionally, the experiments suggest that cell density, that is, how tightly packed together the cells are, plays an important role in controlling how many cells make IFN-I, with a lower cell density resulting in a higher fraction of cells producing IFN-Is. The findings of Van Eyndhoven et al. add to a growing body of evidence reporting heritable states that can guide decision-making in individual cells. Furthermore, it revises our view on how individual immune cells coordinate population-wide responses.

Single-Cell Profiling Reveals Functional Heterogeneity and Serial Killing in Human Peripheral and Ex Vivo-Generated CD34+ Progenitor-Derived Natural Killer Cells.

Increasing evidence suggests that natural killer (NK) cells are composed of distinct functional subsets. This multifunctional role has made them an attractive choice for anticancer immunotherapy. A functional NK cell repertoire is generated through cellular education, resulting in a heterogeneous NK cell population with distinct capabilities responding to different stimuli. The application of a high-throughput droplet-based microfluidic platform allows monitoring of NK cell-target cell interactions at the single-cell level and in real-time. A variable response of single NK cells toward different target cells is observed, and a distinct population of NK cells (serial killers) capable of inducing multiple target lysis is identified. By assessing the cytotoxic dynamics, it is shown that single umbilical cord blood-derived CD34+ hematopoietic progenitor (HPC)-NK cells display superior antitumor cytotoxicity. With an integrated analysis of cytotoxicity and cytokine secretion, it is shown that target cell interactions augment cytotoxic as well as secretory behavior of NK cells. By providing an integrated assessment of NK cell functions by microfluidics, this study paves the way to further functionally characterize NK cells ultimately aimed to improve cancer immunotherapy.

Guidelines for mouse and human DC generation.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

A micropillar array-based microfluidic chip for label-free separation of circulating tumor cells: The best micropillar geometry?

INTRODUCTION: The information derived from the number and characteristics of circulating tumor cells (CTCs), is crucial to ensure appropriate cancer treatment monitoring. Currently, diverse microfluidic platforms have been developed for isolating CTCs from blood, but it remains a challenge to develop a low-cost, practical, and efficient strategy. OBJECTIVES: This study aimed to isolate CTCs from the blood of cancer patients via introducing a new and efficient micropillar array-based microfluidic chip (MPA-Chip), as well as providing prognostic information and monitoring the treatment efficacy in cancer patients. METHODS: We fabricated a microfluidic chip (MPA-Chip) containing arrays of micropillars with different geometries (lozenge, rectangle, circle, and triangle). We conducted numerical simulations to compare velocity and pressure profiles inside the micropillar arrays. Also, we experimentally evaluated the capture efficiency and purity of the geometries using breast and prostate cancer cell lines as well as a blood sample. Moreover, the device's performance was validated on 12 patients with breast cancer (BC) in different states. RESULTS: The lozenge geometry was selected as the most effective and optimized micropillar design for CTCs isolation, providing high capture efficiency (>85 %), purity (>90 %), and viability (97 %). Furthermore, the lozenge MPA-chip was successfully validated by the detection of CTCs from 12 breast cancer (BC) patients, with non-metastatic (median number of 6 CTCs) and metastatic (median number of 25 CTCs) diseases, showing different prognoses. Also, increasing the chemotherapy period resulted in a decrease in the number of captured CTCs from 23 to 7 for the metastatic patient. The MPA-Chip size was only 0.25 cm2 and the throughput of a single chip was 0.5 ml/h, which can be increased by multiple MPA-Chips in parallel. CONCLUSION: The lozenge MPA-Chip presented a novel micropillar geometry for on-chip CTC isolation, detection, and staining, and in the future, the possibilities can be extended to the culture of the CTCs.

A Microfluidic Approach for Probing Heterogeneity in Cytotoxic T-Cells by Cell Pairing in Hydrogel Droplets.

Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific anti-tumoral and anti-viral immunity. When naïve CTLs are activated by antigen-presenting cells (APCs) they display various levels of functional heterogeneity. To investigate this, we developed a single-cell droplet microfluidics platform that allows for deciphering single CTL activation profiles by multi-parameter analysis. We identified and correlated functional heterogeneity based on secretion profiles of IFNγ, TNFα, IL-2, and CD69 and CD25 surface marker expression levels. Furthermore, we strengthened our approach by incorporating low-melting agarose to encapsulate pairs of single CTLs and artificial APCs in hydrogel droplets, thereby preserving spatial information over cell pairs. This approach provides a robust tool for high-throughput and single-cell analysis of CTLs compatible with flow cytometry for subsequent analysis and sorting. The ability to score CTL quality, combined with various potential downstream analyses, could pave the way for the selection of potent CTLs for cell-based therapeutic strategies.

Revising immune cell coordination: Origins and importance of single-cell variation.

Moving from the optimalization of single-cell technologies to the interpretation of the multi-complex single-cell data, the field of immunoengineering is granted with numerous important insights into the coordination of immune cell activation and how to modulate it for therapeutic purposes. However, insights come with additional follow-up questions that challenge our perception on how immune responses are generated and fine-tuned to fight a wide array of pathogens in ever-changing and often unpredictable microenvironments. Are immune responses really either being tightly regulated by molecular determinants, or highly flexible attributed to stochasticity? What exactly makes up the basic rules by which single cells cooperate to establish tissue-level immunity? Taking the type I IFN system and its newest insights as a main example throughout this review, we revise the basic concepts of (single) immune cell coordination, redefine the concepts of noise, stochasticity and determinism, and highlight the importance of single-cell variation in immunology and beyond.

Artificial Antigen-Presenting Cell Topology Dictates T Cell Activation.

Nanosized artificial antigen-presenting cells (aAPCs), synthetic immune cell mimics that aim to activate T cells ex or in vivo, offer an effective alternative to cellular immunotherapies. However, comprehensive studies that delineate the effect of nano-aAPC topology, including nanoparticle morphology and ligand density, are lacking. Here, we systematically studied the topological effects of polymersome-based aAPCs on T cell activation. We employed an aAPC library created from biodegradable poly(ethylene glycol)-block-poly(d,l-lactide) (PEG-PDLLA) polymersomes with spherical or tubular shape and variable sizes, which were functionalized with αCD3 and αCD28 antibodies at controlled densities. Our results indicate that high ligand density leads to enhancement in T cell activation, which can be further augmented by employing polymersomes with larger size. At low ligand density, the effect of both polymersome shape and size was more pronounced, showing that large elongated polymersomes better activate T cells compared to their spherical or smaller counterparts. This study demonstrates the capacity of polymersomes as aAPCs and highlights the role of topology for their rational design.

A universal microfluidic approach for integrated analysis of temporal homocellular and heterocellular signaling and migration dynamics.

Microfluidics offers precise and dynamic control of microenvironments for the study of temporal cellular responses. However, recent research focusing solely on either homocellular (single-cell, population) or heterocellular response may yield insufficient output, which possibly leads to partial comprehension about the underlying mechanisms of signaling events and corresponding cellular behaviors. Here, a universal microfluidic approach is developed for integrated analysis of temporal signaling and cell migration dynamics in multiple cellular contexts (single-cell, population and coculture). This approach allows to confine the desired number or mixture of specific cell sample types in a single device. Precise single cell seeding was achieved manually with bidirectional controllability. Coupled with time-lapse imaging, temporal cellular responses can be observed with single-cell resolution. Using NIH3T3 cells stably expressing signal transducer and activator of transcription 1/2 (STAT1/2) activity biosensors, temporal STAT1/2 activation and cell migration dynamics were explored in isolated single cells, populations and cocultures stimulated with temporal inputs, such as single-pulse and continuous signals of interferon γ (IFNγ) or lipopolysaccharide (LPS). We demonstrate distinct dynamic responses of fibroblasts in different cellular contexts. Our presented approach facilitates a multi-dimensional understanding of STAT signaling and corresponding migration behaviors.

Understanding natural killer cell biology from a single cell perspective.

During the last decade, advances in single cell technologies have ignited increased understanding of natural killer cells (NK cells), which turned out to be far more complex than originally thought. Ample studies have established tissue-specific phenotypic variation within this cell population; however, the functional implication of this vast variation is still unclear. At single-cell level, the function of a NK cell is tightly regulated by several checkpoints however upon proper recognition the cell can deliver a lytic hit as early as 10 min or could take hours before they can kill their target cells. Moreover, only a fraction of NK cells appears to kill target cells while the larger portion of NK cells appear to be non-cytotoxic. All these studies showed that the NK cell compartment is composed of cells with different functional strengths and efficacies, thereby highlighting the necessity of analytical platforms that allow the study of these important innate immune cells at single-cell level. In this review, we discuss and provide an overview on phenotypical and functional heterogeneity within the NK cell population and subsequently provide information regarding emerging technologies that highlight the importance of single-cell studies to understand the biology of these cells.

Robust Antigen-Specific T Cell Activation within Injectable 3D Synthetic Nanovaccine Depots.

Synthetic cancer vaccines may boost anticancer immune responses by co-delivering tumor antigens and adjuvants to dendritic cells (DCs). The accessibility of cancer vaccines to DCs and thereby the delivery efficiency of antigenic material greatly depends on the vaccine platform that is used. Three-dimensional scaffolds have been developed to deliver antigens and adjuvants locally in an immunostimulatory environment to DCs to enable sustained availability. However, current systems have little control over the release profiles of the cargo that is incorporated and are often characterized by an initial high-burst release. Here, an alternative system is designed that co-delivers antigens and adjuvants to DCs through cargo-loaded nanoparticles (NPs) incorporated within biomaterial-based scaffolds. This creates a programmable system with the potential for controlled delivery of their cargo to DCs. Cargo-loaded poly(d,l-lactic-co-glycolic acid) NPs are entrapped within the polymer walls of alginate cryogels with high efficiency while retaining the favorable physical properties of cryogels, including syringe injection. DCs cultured within these NP-loaded scaffolds acquire strong antigen-specific T cell-activating capabilities. These findings demonstrate that introduction of NPs into the walls of macroporous alginate cryogels creates a fully synthetic immunostimulatory niche that stimulates DCs and evokes strong antigen-specific T cell responses.

Decoding the dynamics of multilayered stochastic antiviral IFN-I responses.

Type I Interferon (IFN-I) responses were first recognized for their role in antiviral immunity, but it is now widely appreciated that IFN-Is have many immunomodulatory functions, influencing antitumor responses, autoimmune manifestations, and antimicrobial defenses. Given these pivotal roles, it may be surprising that multilayered stochastic events create highly heterogeneous, but tightly regulated, all-or-nothing cellular decisions. Recently, mathematical models have provided crucial insights into the stochastic nature of antiviral IFN-I responses, which we critically evaluate in this review. In this context, we emphasize the need for innovative single-cell technologies combined with mathematical models to further reveal, understand, and predict the complexity of the IFN-I system in physiological and pathological conditions that may be relevant to a plethora of diseases.

An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip.

Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.

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

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.

Programmable Bivalent Peptide-DNA Locks for pH-Based Control of Antibody Activity.

The ability to control antibody activity by pH has important applications in diagnostics, therapeutic antibody targeting, and antibody-guided imaging. Here, we report the rational design of bivalent peptide-DNA ligands that allow pH-dependent control of antibody activity. Our strategy uses a pH-responsive DNA triple helix to control switching from a tight-binding bivalent peptide-DNA lock into a weaker-binding monovalent ligand. Different designs are introduced that allow antibody activation at both basic and acidic pHs, either autonomously or in the presence of an additional oligonucleotide trigger. The pH of antibody activation could be precisely tuned by changing the DNA triple helix sequence. The peptide-DNA locks allowed pH-dependent antibody targeting of tumor cells both in bulk and for single cells confined in water-in-oil microdroplets. The latter approach enables high-throughput antibody-mediated detection of single tumor cells based on their distinctive metabolic activity.

A Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms.

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and analyzing cells at the single-cell level by eliminating the influence of external factors on the cellular microenvironment. Encapsulation of cells in droplets is dictated by the Poisson distribution as a function of the number of cells present in each droplet and the average number of cells per volume of droplet. Primary cells, especially immune cells, or clinical specimens can be scarce and loss-less encapsulation of cells remains challenging. In this paper, we present a new methodology that uses pipette-tips to load cells to droplet-based microfluidic devices without the significant loss of cells. With various cell types , we demonstrate efficient cell encapsulation in droplets that closely corresponds to the encapsulation efficiency predicted by the Poisson distribution. Our method ensures loss-less loading of cells to microfluidic platforms and can be easily adapted for downstream single cell analysis, e.g., to decode cellular interactions between different cell types.

Development of Morphologically Discrete PEG-PDLLA Nanotubes for Precision Nanomedicine.

Precise control over the morphological features of nanoparticles is an important requisite for their application in nanomedical research. Parameters such as size and shape have been identified as critical features for effective nanotherapeutic technologies due to their role in circulation, distribution, and internalization in vivo. Tubular PEG-PDLLA polymersomes (nanotubes) exhibit an interesting morphology with potential for immunotherapeutics, as the elongated shape can affect cell-particle interactions. Developing methodologies that permit control over the precise form of such nanotubes is important for their biomedical implementation due to the stringent physicochemical constraints for efficacious performance. Through careful control over the engineering process, we demonstrate the generation of well-defined nanotubes based on polymersomes as small as 250 and 100 nm, which can be successfully shape transformed. The quality of the resulting nanostructures was established by physical characterization using AF4-MALS and cryo-TEM. Moreover, we show the successful loading of such nanotubes with model payloads (proteins and drugs). These findings provide a promising platform for implementation in biomedical applications in which discrete structure and functionality are essential features.