The adaptive immune system is finely tuned by diverse highly specific ligand-receptor interactions. Being able to decode these interactions at the single-cell level will further our understanding of the complexities of the immune response.

To this end, researchers at Stanford University led by Howard Chang have developed ENTER, a method that leverages lentiviral-mediated cell entry to deorphanize ligand–receptor pairs. First the team engineered lentiviruses expressing a mutant vesicular stomatitis virus protein G (VSV-G) that allows them to bind low-density lipoprotein receptors on host cells without infecting them. When a ligand of interest is co-expressed with the VSV-G, the viruses specifically binds the cells expressing the cognate receptor, allowing these cells to be isolated and further analyzed.

This VSV-G modular viral display system for deciphering ligand–receptor pairing has enabled high-throughput, large-scale T cell receptor (TCR) and B cell receptor (BCR) profiling (Dobson, C.S. et al. Nat. Methods 19, 449–460; 2022).

Similarly, researchers have now engineered viruses to display a single-chain antigen–MHCI (major histocompatibility complex I) to identify the cognate TCRs. This approach was then extended to B cells, where an optimized transmembrane domain was engineered for the surface display of B cell antigens. For example, viruses were engineered to display the receptor-binding domain from SARS-CoV-2 and could be used to detect cells expressing the cognate BCR with high specificity.

Next, the researchers demonstrated that ENTER could be used to deliver cargo to T or B cells in an antigen-specific manner. When they delivered the GFP transgene, 82% of the target T cells and only 0.22% of non-target T cells expressed the fluorescent protein. As a proof of concept, the team engineered peptide–MHC-displaying viruses to deliver a suicide gene to antigen-specific T cells for targeted cell killing.

To further extend the applications of this approach, the researchers then combined ENTER with a droplet-based single-cell RNA-sequencing pipeline. This method, called ENTER-seq, could simultaneously map antigen specificity, TCR repertoire and single T cell gene expression profiles. Using ENTER-seq, the team delved into the memory T cell repertoire in cytomegalovirus seropositive individuals.

ENTER and ENTER-seq are versatile methods not only for deorphanizing immune ligand–receptor pairs but also for high-content screening of single T cells.

Original reference: Cell 185, 4904–4920.e22 (2022)