The use of engineered immune cell-based therapies in treating hematologic malignancies has shown exciting results with improved patient outcomes including complete remissions, however, there are still significant variations in outcomes between patients and clinical trials. The biological basis behind these variations is still not well understood, but a growing body of literature has alluded to certain T-cell functional properties such as high proliferative potential and propensity to secrete multiple cytokines simultaneously as key drivers of response. However, tools to analyze and select populations of cells based on functional properties, such as secreted products, are critically lacking. Current immune cell phenotyping approaches are limited by heavy reliance on screening differentially expressed surface receptors which fails to describe their functional potency. Therefore, there is a critical need to develop new platforms to sort based on cell function, such as level and types of secreted cytokines to identify gene expression signatures associated with functional responses, uncover cell surface markers that are more descriptive of optimal functional phenotypes, or directly sort starting cell populations with higher therapeutic potential during manufacturing of cell therapy.
Here, we report a workflow for the rapid screening and sorting of individual T-cells based on secreted factors that are accumulated on 3D-structured microparticles using a standard fluorescence activated cell sorter (FACS). Our cavity-containing hydrogel microparticles (nanovials) can be loaded with single T-cells and encapsulated into uniform droplets by simple pipetting steps to confine secreted cytokines for capture on the particle. Captured cytokines (TNF-α and IFN-γ) are each labelled with a fluorescent reporter antibody, and the corresponding cells are sorted based on the secretion level using a commercial FACS. Improving on our previous work to adhere Chinese Hamster Ovary cells and capture secreted antibodies on nanovials, we have further modified the nanovials to be optimized for T-cell cytokine secretion assay by incorporating anti-CD45 antibody as a new binding moiety on their surface and reducing their size down to 35 μm in diameter for improved cell loading efficiency and high-throughput sorting.
We have demonstrated our ability to capture and label TNF-α, IFN-γ secretions produced from single T-cells and sorted nanovials containing cells using fluorescence height and area signals above a threshold value. By gating on a combination of fluorescence area and height parameters, we can differentiate secreted cytokine signal on nanovials from signal solely from presumably permeabilized or dead cells. Cells also maintained their viability after sorting, in which they can be further analyzed for their growth and functionality post-sort. Our technology can quantitatively screen and sort potentially millions of viable cells based on their cytokine secretion levels, which will aid in discovery of T-cell receptors and surface markers responsible for robust functional responses, improving upon the current paradigm of cell therapy.
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