IL-2 secretion-based sorting of single T cells using high-throughput microfluidic on-cell cytokine capture

Abstract

Secreted proteins are critical for the coordination of potent immune defenses, such as in engineered T cell therapies, however, there are few widely accessible approaches to accurately analyze and sort large numbers of cells based on their secretory functions. We report a workflow for the rapid screening and sorting of single individual T cells based on IL-2 secretion accumulated at high concentrations in nanoliter droplets and encoded back onto the secreting cell's surface. In our method, droplets are used solely to partition cells, enabling rapid accumulation of signals onto cell surfaces, and eliminating diffusive crosstalk between neighbors. All downstream sorting leverages conventional high-throughput and readily accessible flow cytometry after the emulsion is disrupted. We achieve monodisperse droplet generation (CV < 10%) at flow rates up to 200 μL min⁻¹ using step emulsification, enabling processing of entire libraries of cells within tens of minutes without significant secretion crosstalk. In comparison to our approach, strong mitogenic activation overwhelmed the conventional bulk on-cell cytokine assay, rendering labeled, non-activated cells indistinguishable from actively secreting neighbors within one hour. Processing of identical cell mixtures following droplet encapsulation yielded no apparent crosstalk even after three hours. Instead, IL-2 production spanning several orders of magnitude was observed from roughly 20% of analyzed activated lymphocytes, representing an at least 10-fold increase in dynamic range compared to unencapsulated cells. Secreting cells could also be sorted using fluorescence activated cell sorting (FACS). The approach can ultimately enable sorting of cells based on functional properties with higher accuracy in a more accessible format to life science researchers.