Poster CC17 - Characterizing T Cell Functional Properties on a Single Cell Scale Using a Hydrogel-Based Platform

Introduction: Technology to analyze single-cell-based secretions has the potential to deepen our understanding of cellular biology. This is most applicable in engineered cell therapies, especially chimeric antigen receptor (CAR) T cell therapies, wherein identifying particular T cell functional properties can be key to understanding response and efficacy. However, secreted molecules rapidly diffuse away from cells, and analysis of these secretions requires specialized equipment and expertise to analyze. A functional assay at a single-cell level was developed using hydrogel-based chemically functionalized microcontainers, denoted as nanovials (Figure 1). These nanovials have been shown to effectively load and stimulate single, viable, cells that can be sorted based on their secreted products using standard florescence-activated sorting (FACS) technologies. We show that this technology can be extended to capture, sort, and characterize mouse T cells with the goal of better understanding secretions key to immune cell function.

Materials and

Methods: Using nanovial technology, we characterized the secretion of interferon-gamma (IFN-γ) by individual CD8+ T cells harvested from OT-I mice. OT-I mice spleen and lymph nodes were harvested and processed to isolate CD8+ T cells. After an initial expansion, the T cells were stained and loaded into functionalized nanovials where they are specifically activated with ovalbumin (OVA) peptide. The cell-loaded nanovials were then sorted based on cytokine secretion levels via FACS to isolate various cell subsets based on secretion of IFN-g as a readout for T cell receptor signaling.

Results, Conclusions, and Discussions: We have successfully leveraged the nanovial platform for mouse CD8+ T cell loading and sorted cell-loaded particles based on cytokine secretion. Unlike traditional cytokine secretion assays which require fixation and permeabilization, cells in nanovials remain viable throughout this process, allowing for sorted cells to be further analyzed and manipulated. Ongoing and future work includes cell analysis post FACS sorting and analysis of additional T cell subsets as well as other immune cell lineages. Collectively, our efforts introduce a new paradigm for elucidating the functional properties of immune cells, providing critical insights that will guide the future design of engineered cell therapeutics.

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