ENTER-seq – multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells

Cells communicate with each other via receptor-ligand interactions. Researchers at Stanford University have developed lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. They optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. The researchers engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

ENTER-seq for massively parallel measurement of
antigen peptide sequence, TCR sequence, and transcriptome

(A) Schematic view of ENTER-seq workflow. A library of pooled viruses displaying individual pMHC was incubated with T cells for 2 h. GFP+ T cells were sorted for droplet-based single-cell genomics profiling. (B) Viral RNA engineering strategy for droplet-based single-cell capture. 10X Genomics capture tag is inserted in the linker region between B2M and HLA-A2. 10X Genomics PCR handle is inserted after CMV promoter. CMV, CMV promoter; SP, signal peptide sequence; peptide, antigen peptide; B2M, beta-2-microglobulin; MHC class I, HLA-A0201 allele; LTR, long terminal repeat; TSO, template-switching oligo sequence. (C) Schematic view of T cell mixing experiment for ENTER-seq. (D) Number of pp65495–503-TCR T cells UMI counts (x axis) and NY-ESO-1157–165-TCR UMI counts (y axis) associated with each cell barcode (dot). The colors are assigned as NY-ESO-1157–165-TCR+ T cells (light blue), pp65495–503-TCR+ T cells (red), and doublets (green, with both NY-ESO-1157–165-TCR and pp65495–503-TCR). (E) Scatterplots of TCR UMI counts after doublet removal, colored by enrichment ratio of pp65495–503-antigen UMI count among total UMI counts (left) and enrichment ratio of NY-ESO-1157–165-antigen UMI count among total UMI counts (right). (F) Number of pp65(495–503)-antigen UMI counts (x axis) and NY-ESO-1157–165-antigen UMI counts (y axis) associated with each cell barcode (dot) after doublet removal. The colors are assigned as NY-ESO-1157–165-TCR+ T cells (light blue) and pp65495–503-TCR+ T cells (red).