Sorting single T cells based on secreted cytokines and surface markers using hydrogel nanovials

Abstract

Immune cell function is intrinsically linked to secreted factors which enable cells to communicate with neighboring or distant cells to coordinate a response. The ability to secrete cytokines also can help define the population of cells with therapeutic potential in emerging cell therapies, such as chimeric antigen receptor (CAR)-T cell therapies. Polyfunctional cells that can secrete more than one cytokine have been found to play an outsized role in therapeutic efficacy. While there are a variety of techniques to analyze cellular secretions from individual polyfunctional cells, there are no widely-available approaches to sort viable cells based on this phenotype. Here, we apply lab on a particle technology to the analysis and sorting of T cells based on a combination of secreted factors, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and interleukin 2 (IL-2) and surface markers (CD8+ and CD4+). Cells are selectively loaded into the antibody-functionalized cavity of micro-hydrogel particles, called nanovials, where secreted cytokines are captured and fluorescently stained. By leveraging standard fluorescence activated cell sorters and using fluorescence pulse area/height information we can distinguish between fluorescence signals on the nanovial cavities and on cells, and are able to process greater than 1 million nanovials in one hour of sorting. The frequency of multi-cytokine secreting cells was correlated with surface marker expression, and biased towards CD4+ T cells. CD8+ cells that secreted more than one cytokine, were biased towards IFN-γ and TNF-α with fewer CD8+ cells secreting IL-2. The majority of cells with a polyfunctional phenotype that were sorted remained viable and regrew following sorting. This nanovial cytokine secretion assay can be applied to sort antigen-specific T cells or CAR-T cells based on their functional engagement with cognate antigens or peptide-major histocompatibility complexs (MHCs), enabling discovery of functional CARs or T cell receptors and deeper investigation into the molecular underpinnings of single T cell function.

Defining T cell receptor repertoires using nanovial-based affinity and functional screening

“…Polyethylene glycol biotinylated nanovials with 35 μm diameters were fabricated using a three-inlet flow-focusing microfluidic droplet generator, sterilized and stored at 4°C in Washing Buffer consisting of Dulbecco’s Phosphate Buffered Saline (Thermo Fisher) with 0.05% Pluronic F-127 (Sigma), 1% 1X antibiotic-antimycotic (Thermo Fisher), and 0.5% bovine serum albumin (Sigma) as previously reported ( 30 ).…”

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

“…Secretions from cells within the nanovials can be detected by staining with fluorescently labeled antibodies and sorted based on fluorescent signal. This approach has been demonstrated in previous work characterizing secreted antibodies from B cells, Chinese Hamster Ovary cells 10,12 , and of cytokines from T cells 13 . Overall, our findings demonstrate the need to probe secretion and transcriptome simultaneously to better understand the regulation of protein secretion.…”

Lab on a Particle Technologies

“…Secreted analytes captured on the nanovial are then stained with fluorescently labeled and/or oligonucleotide-labeled detection antibodies (Figure d). Multiple capture antibodies against different secretions can also be immobilized on a single nanovial for multiplexed analysis …”