Dynamic analysis of immune and cancer cell interactions at single cell level in microfluidic droplets

Abstract

Cell-cell communication mediates immune responses to physiological stimuli at local and systemic levels. Intercellular communication occurs via a direct contact between cells as well as by secretory contact-independent mechanisms. However, there are few existing methods that allow quantitative resolution of contact-dependent and independent cellular processes in a rapid, precisely controlled, and dynamic format. This study utilizes a high-throughput microfluidic droplet array platform to analyze cell-cell interaction and effector functions at single cell level. Controlled encapsulation of distinct heterotypic cell pairs was achieved in a single-step cell loading process. Dynamic analysis of dendritic cell (DC)-T cell interactions demonstrated marked heterogeneity in the type of contact and duration. Non-stimulated DCs and T cells interacted less frequently and more transiently while antigen and chemokine-loaded DCs and T cells depicted highly stable interactions in addition to transient and sequential contact. The effector function of CD8⁺ T cells was assessed via cytolysis of multiple myeloma cell line. Variable cell conjugation periods and killing time were detected irrespective of the activation of T cells, although activated T cells delivered significantly higher cytotoxicity. T cell alloreactivity against the target cells was partially mediated by secretion of interferon gamma, which was abrogated by the addition of a neutralizing antibody. These results suggest that the droplet array-based microfluidic platform is a powerful technique for dynamic phenotypic screening and potentially applicable for evaluation of novel cell-based immunotherapeutic agents.

FIG. 1.

Cell co-encapsulation in droplet microfluidic platform. (a) Schematic of integrated droplet generation and a microarray device. (b) Generation of nanoliter droplets. (c) Droplets loaded in a microarray for stable docking. (d) Morphology of single DC and T cell in the droplet. Inset: Magnified image of dendrite extension by DC. (e) Cellular exocytosis observed in the droplet. Inset: Magnified image of vesicles secreted by DC at 4 h. Scale bar: 20 μm. (F) Representative mean velocity profiles of one T cell, DC, and RPMI-8226 cell in the droplet. (g) A decrease in velocity as cells near each other and form conjugate. The onset of contact period is indicated by ♦. RPMI-8226 cell death occurred at 240 min and the complex dissociated rapidly.

FIG. 2.

Dynamic monitoring of interaction between stimulated DC and CD3+ T cells in microfluidic droplets. (a) DCs were pulsed with OVA-FITC (100 μg/ml, 16 h) and CCL21 (25 ng/ml, 2 h) and co-encapsulated with untreated T cells in droplets. OVA-FITC expression on the DC surface is indicated by arrowheads. T cells are labeled with CMTPX tracker (red), which is transferred to the DCs over time. A series of time-lapse images of the same droplet is shown over a period of 1 h. Images were obtained every 5 min. Scale bar: 20 μm. (b) Analysis of the types of interaction between DC and T cell: no interaction over a period of 5 h (-), continuous interaction for 5 h and discontinuous interaction defined by short periods of attachment and detachment. DCs were pre-treated with OVA-FITC and CCL21 (n = 227 cell pairs) or untreated (n = 100). (c) Cells undergoing a discontinuous interaction were further categorized into transient (≤10 min of contact) and stable (≥10min) interactions. (d) Distribution of contact times between DC and T cells (outliers are indicated). The data are represented as mean ± SEM of n = 3 independent experiments. P < 0.05 is indicated by *.

FIG. 3.

(a) Interaction between multiple cells in droplets. DCs (green) were treated with OVA-FITC and CCL21. The CD3+ T cells were untreated and labeled with CMTPX (red) tracker. The panels (phase, fluorescent, and overlay) depict DC and T cells at various time points (representative of n = 30 droplets). Scale bar: 20 μm. (b) Number of contacts made between treated DC-T cell pairs (1:1 ratio). The data are represented as mean ± SEM from three independent experiments.

FIG. 4.

Interaction between CD8+ T cells and target cells (RPMI-8226) in droplets. (a) Lysis and death of target cell in contact with CD8+ T cell. Target cells (green) were labeled with Calcein AM. Scale bar: 50 μm. The T cells were not labeled. (b) % of target cell death mediated by T cells (n = 3, mean ± SEM). P < 0.05 is indicated by *. A total of n = 292, 210, and 144 cells were quantified in Control, PMA/Ionomycin-treated, and PMA/Ionomycin/IFN-γ-treatment groups, respectively. (c) Comparison of time required by PMA/Ionomycin-treated T cells to promote target cell death. A threshold of 200 min was set for distinguishing fast vs. slow kill. (d) Effect of the presence/absence of IFN-γ antibody on rapid target death. Cell numbers (n) used for analysis in this representative experiment are indicated. (e) Number of contacts made between activated T cell-target cell pairs in a representative experiment. n: 104, 98, and 53 for Control, PMA/Ionomycin-treated, and PMA/Ionomycin/IFN-γ-treated cells, respectively.