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

**Bart M. Tiemeijer 1,2 , Lucie Descamps 1,2 , Jesse Hulleman 1,2, Jelle J. F. Sleeboom <sup>3</sup> and Jurjen Tel 1,2,\***


**Abstract:** Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific antitumoral 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 singlecell 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.

**Keywords:** single-cell; CD8 T-cell; cytokines; droplet microfluidics; microgel; heterogeneity
