Detection of cell–cell interactions via photocatalytic cell tagging

Abstract

Cell-cell interactions drive essential biological processes critical to cell and tissue development, function, pathology, and disease outcome. The growing appreciation of immune cell interactions within disease environments has led to significant efforts to develop protein-and cell-based therapeutic strategies. A better understanding of these cell-cell interactions will enable the development of effective immunotherapies. However, characterizing these complex cellular interactions at molecular resolution in their native biological contexts remains challenging. To address this, we introduce photocatalytic cell tagging (PhoTag), a modality agnostic platform for profiling cell-cell interactions. Using photoactivatable flavin-based cofactors, we generate phenoxy radical tags for targeted labeling at the cell surface. Through various targeting modalities (e.g. MHC-Multimer, antibody, single domain antibody (VHH)) we deliver a flavin photocatalyst for cell tagging within monoculture, co-culture, and peripheral blood mononuclear cells. PhoTag enables highly selective tagging of the immune synapse between an immune cell and an antigen-presenting cell through targeted labeling at the cell-cell junction. This allowed for the ability to profile gene expression-level differences between interacting and bystander cell populations. Given the modality agnostic and spatio-temporal nature of PhoTag, we envision its broad utilization to detect and profile intercellular interactions within an immune synapse and other confined cellular regions for any biological system..

Proteomic mapping of intercellular synaptic environmentsviaflavin-dependent photoredox catalysis

“…3b, middle and bottom panel). These results are consistent with previous observations of promiscuous labeling that occurs inside and outside of the synaptic region using HRP 24 and as further confirmed by confocal imaging within this JY-Jurkat co-culture system (ESI Fig. 9 †).…”

“…1d). 24 Accordingly, we site-selectively functionalized a blocking α-PD-L1 VHH-Fc that disrupts the PD-1/PD-L1 interaction with riboflavin tetraacetate (RFT) (ESI Fig. 1a†) for PD-L1-targeted proximity labeling.…”

“…Enrichment of this cis interactor resulted from the use of the non-blocking VHH that, in addition to lacking PD-1/ PD-L1 blocking capacity, does not block PD-L1/CD80 interactions. 24 Differences in protein enrichment between blocking and non-blocking α-PD-L1 VHH-Fc targeting modalities were analyzed using a quadrant plot that compares the fold change enrichment for statistically significantly enriched proteins identified in blocking and/or non-blocking proximity labeling experiments (Fig. 2c).…”

“…[20][21][22][23] Photocatalytic-based approaches are also expanding into more complex cellular environments as demonstrated through our recently disclosed photocatalytic cell tagging (PhoTag) technology. 24 This method exploits visible light-activation of flavin co-factors to generate phenoxy radicals 21 (Fig. 1c) for selective tagging of physically associated cellular interfaces to facilitate downstream transcriptome analysis of cell-cell interactions.…”

Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling

“…Proximity labeling has been widely investigated in recent years and is used to analyze protein–protein interactions in living cells [ 13 , 14 ], cell membranes [ 15 , 16 ], ligand recognition on cell membrane surfaces [ 17 , 18 ], and intercellular communication [ 19 ]. APEX and BioID are performed to chemically label proteins in proximity to a target protein to which an enzyme is fused [ 20 , 21 ].…”