cloneXplorer: A high-throughput clone discovery platform based on conical microwell arrays

Abstract

Antigen-specific T cell populations are of great value for studying immune recognition but tedious to generate by limiting dilution or cloning. Here, we develop a streamlined approach to generate antigen-specific T cell clones directly from peripheral blood using the cloneXplorer, a live-cell analysis and clone isolation platform based on conical microwell arrays. This platform continuously monitors cell proliferation, cytokine secretion, and surface markers in up to 100,000 single cell co-cultures, enabling the identification of rare, functionally defined T cells, which can be recovered for clonal expansion or sequence analysis. We benchmark the platform by performing several key demonstrations. First, we show that this platform can efficiently generate monoclonal cell populations from cell lines and human T cells. Next, we demonstrate that antigen-specificity can be identified at single cell resolution using a co-culture of Jurkat cells expressing NFAT-GFP, CD8, and a T cell receptor and K562 antigen presenting cells (APC) expressing a peptide library. Thereafter, we show that immune activation in mouse and human primary samples can be monitored by time lapse analysis of Interferon gamma (IFN-{gamma}) secretion in individual microwell co-cultures using a fluorescent sandwich assay. Finally, we combine these capabilities in a proof-of-concept demonstration, which uses IFN-{gamma} secretion and the presence of CD8 surface markers as hierarchical gates to isolate and expand antigen-specific T cells from human peripheral blood, and we verify their specificity by tetramer staining. Together, these results showcase potential applications of the cloneXplorer platform in cell line development, and in screening and validating immune receptor interactions with specific antigens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/699323v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1aafc29org.highwire.dtl.DTLVardef@91272dorg.highwire.dtl.DTLVardef@1a306eeorg.highwire.dtl.DTLVardef@1bfd54_HPS_FORMAT_FIGEXP M_FIG C_FIG