Preprint Club
A cross-institutional Journal Club Initiative

Keywords

·  Cellular interaction

·  Cell contact

·  Proximity-labeling

Main Findings

Cell-to-cell interaction defines cellular function in tissues. However, the available tools for reliably monitoring and tracking these interactions in vivo are limited. Proximity-based labeling has emerged as a robust method for identifying cellular contacts by employing enzymatic transfer of chemical substrates between cells. A technique called LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts) was previously developed to detect cellular interactions between dendritic and T cells in vivo driven by CD40L-CD40 receptor-ligand pair.

In the recent preprint, the research team has further advanced this technique by transforming the original LIPSTIC into a universal system called uLIPSTIC. This updated system enables unbiased recording of the cellular interactions between any cell subsets, without being limited to immune cells or a specific receptor/ligand.

Generating a mouse allele carrying the modified uLIPSTIC construct, authors successfully identified the interaction partners of various cell types in vivo, including CD4, CD8, and regulatory T cells, as well as different dendritic cell subsets and germinal center B cells. Taking an innovative approach, the team then combined uLIPSTIC with a CITE-seq single-cell transcriptomics workflow and uncovered the maturation dynamics of CD4 T cells in the intraepithelial compartment of the gut using interaction-based transcriptomics.

Limitations

Although the study is very well executed, authors could consider addressing the following points:

·  Substrate stability and turnover at acceptor sites are important factors that determine the temporal kinetics and dynamic range of labeling. However, these properties have not been critically evaluated and reported in the uLIPSTIC system.

·  For validation purposes, the authors investigated cellular compartments using uLIPSTIC where interaction dynamics are relatively well-established. While the findings from the intraepithelial compartment of the gut are highly novel and interesting, applying uLIPSTIC to an additional compartment with a less –well known interactome could substantially increase the novelty and significance of the uLIPSTIC system, providing more diversification from the original LIPSTIC paper.

·  Interactions between cells within the same subset have important regulatory roles. Despite the improved range of applicability of uLIPSTIC, it currently stands at ‘universal-1’ subset coverage, as it is not capable of recording cellular interactions between the same cell types due to the mutually exclusive expression of acceptor and donor domains. However, adoptive transfer approaches with congenic Cre-negative acceptor cells matching the donor cell type could overcome this limitation.

·  Although the detection of uLIPSTIC labeling via single-cell RNA-seq and/or flow cytometry is a significant improvement, the compatibility of uLIPSTIC with microscopy techniques or spatial transcriptomics has not been tested or discussed in the study. Additionally, while the authors have rigorously tested that uLIPSTIC labeling is dependent on functional interactions, it is important to consider the possibility that some of these contacts may have been artificially introduced during tissue processing (e.g., digestion) when cells were liberated from their niches. Therefore, validating the spatial accuracy of interactions captured by uLIPSTIC through imaging-based methods would be valuable, particularly for complex tissues.

·  As acknowledged by the authors in the discussion, uLIPSTIC is a genetically engineered construct that requires cell-type-specific Cre-recombinase expression to operate. Thus, the system is limited by the availability and robustness of the Cre transgene. Furthermore, it remains unclear how applicable uLIPSTIC is to human model systems.

Significance/Novelty

The uLIPSTIC system offers several significant advantages. Its distinctive features, such as the very low affinity between acceptor and donor sites and continuous non-binary recording capacity, allow for highly specific detection of cellular interactions in a quantitative manner that cannot be achieved by other conventional methods.

Moreover, uLIPSTIC can be coupled to single-cell RNA-seq methods to generate an interactome atlas with an unprecedented dynamic range and accuracy. Unlike computational workflows, uLIPSTIC does not rely on doublet formation between cells and subsequent deconvolution. Additionally, its receptor-ligand agnostic property enables unbiased exploration of cell-to-cell interaction in vivo.

Overall, uLIPSTIC paves the way for studying intercellular communication in vivo, extending beyond immune cells and enabling a more complete understanding of cellular dynamics across various cell subsets and tissues.

Credit

Reviewed by Yavuz Yazicioglu as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.