Rajaei, H. | bioRxiv Science

bioRxiv2026

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

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

cell biology↗

bioRxiv2021

Adar1 deletion causes degeneration of exocrine pancreas via Mavs-dependent interferon signaling

Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-binding protein that deaminates adenosine(A) to inosine(I). A-to-I editing alters post-transcriptional RNA processing making ADAR1 a critical regulator of gene expression. Consequently, Adar1 has been implicated in organogenesis. To determine the role of Adar1 in pancreatic development and homeostasis, we specifically deleted Adar1 from the murine pancreas (Ptf1aCre/+; Adar1Fl/Fl). The resulting mice had stunted growth likely due to malabsorption associated with exocrine pancreas insufficiency. Analyses of pancreases revealed ductal expansion, heightened interferon-stimulated gene expression and an increased influx of immune cells. In addition, we observed an increased prevalence of CD4+ T and natural killer cells in their splenic tissue. These results indicate an association between loss of pancreatic Adar1 with dysregulation of systemic immunity. Concurrent deletion of Adar1 and Mavs, a signaling protein implicated in the innate immune pathway rescued the degenerative phenotype and resulted in normal pancreatic development. Taken together, our work suggests that the primary function of Adar1 in the pancreas is to prevent aberrant activation of the Mavs-mediated innate immune pathway, thereby maintaining pancreatic homeostasis. Summary statementThis work defines the role of Adar1 in pancreatic development and homeostasis.

developmental biology↗