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

Abstract

The ability to selectively bind to antigenic peptides and secrete cytokines can define populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with millions of peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs and secrete cytokines on nanovials, allowing sorting based on both affinity and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αβ-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes we could link TCR sequence to targets with 100% accuracy. We identified with high specificity an expanded repertoire of functional TCRs targeting viral antigens compared to standard techniques.

Optimizing Cell Therapy by Sorting Cells with High Extracellular Vesicle Secretion

“…For example, multiple detection antibodies could be applied with different fluorophores and/or the nanovial surface could be conjugated with more than one capture antibody targeting different EV-surface presented proteins, including engineered targeting protein 29 . We have previously shown the ability to multiplex the detection of secreted cytokines 30 . By further characterizing the types of EVs released, researchers could further sub-categorize cells based on the propensity to secrete subsets of EVs and refine our understanding of what are the most important sets of EVs to be released for therapeutic benefit.…”

Linking single-cell transcriptomes with secretion using secretion-encoded single-cell sequencing (SEC-seq)

“…To link the amount of secretion of a target protein from a cell with the same cell's transcriptome we have developed Secretion EnCoded single-cell sequencing, or SEC-seq [3][4][5] . As a first step, we needed to devise a method by which the secretions from a single cell could be spatially associated with it in a downstream transcriptomic workflow.…”

“…Another powerful example of SEC-seq was its use in characterizing T cells isolated by their T-cell receptor (TCR) specificity. T cells expressing TCRs that recognize specific antigenic peptides presented by major histocompatibility complex I (pMHC) could be isolated by linking the pMHC molecules on Nanovials and then characterizing the correlated secretion of effector molecules triggered for secretion from the specifically bound T cells 5 (Figure 2c). Captured cells were then interrogated by SEC-seq for granzyme B secretion, which was linked to both their transcriptomes and their specific TCR.…”

“…SEC-seq can probe biomolecules secreted at high rates (like IgG) or low rates (like VEGF-A) by adjusting the time allotted for secretion accumulation. Secretions induced by cell-cell interactions can also be analyzed, as demonstrated with T cells secreting cytokines in response to interaction with pMHC molecules bound to the Nanovial surface 5 . Finally, we anticipate other techniques, like functional approaches such as Perturb-seq can be combined with SEC-seq to delve into the network of genes controlling secretion of various factors.…”

“…Using a secondary oligo-barcoded detection antibody against a primary detection antibody is also a viable strategy to obtain readily available reagents for your target secretion. For example, in the T-cell study 5 , granzyme B was targeted with by an APC-conjugated antibody, and detection was mediated by a secondary oligo-barcoded anti-APC antibody.…”