Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node
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Abstract
The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a ‘molecular tracking device’ to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 26 2020, follows.
Summary
The idea of using DNA tags to enable tracking of protein and its subsequent handling is innovative and interesting. The manuscript is well written and some of the notions presented may help drive the field forward. There is a potential to revise the manuscript for eLife as a proof of principle for the approach of using scRNA seq to track an antigen in vivo.
Essential Revisions
While the archiving of the psDNA-Ova conjugate in lymphatic endothelial cells matches the earlier observations of fluorescently tagged Ova, the power of this method is to work with scRNAseq to be able to identify novel cells that interact the the complex. In this regard, it's clear that the psDNA-Ova may interact differently …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 26 2020, follows.
Summary
The idea of using DNA tags to enable tracking of protein and its subsequent handling is innovative and interesting. The manuscript is well written and some of the notions presented may help drive the field forward. There is a potential to revise the manuscript for eLife as a proof of principle for the approach of using scRNA seq to track an antigen in vivo.
Essential Revisions
While the archiving of the psDNA-Ova conjugate in lymphatic endothelial cells matches the earlier observations of fluorescently tagged Ova, the power of this method is to work with scRNAseq to be able to identify novel cells that interact the the complex. In this regard, it's clear that the psDNA-Ova may interact differently with various cell types due to the potential for recognition of the psDNA, particularly by TLR9 as suggested. Tracking the psDNA-Ova conjugate as an immunogenic adjuvant-antigen complex is an interesting starting point. You can address this concern by reframing the goal from tracking a protein antigen to characterizing the archiving and presentation of the barcoded psDNA-Ova complex. This doesn't require any additional work, just changing the way you set it up- that this if you immunogen is a DNA-protein complex- you can track it by scRNA-seq. The potential to study trafficking in a TLR9 KO mice in the future might open up using this method more generically for tracking the protein antigen, but this would require much more work to rule out other influence of the DNA on archiving and processing of the antigen.
Your evidence that the psDNA really reflects the distribution of the protein is not sufficient. In Figure 1 c and d it's not clear how you measure the amount of protein? Is this the protein injected or the protein detected in a immunoblot or capture immunoassay? To extend the in vitro analysis in Figure 1c and d to actually visualize the native protein with anti-Ova and the psDNA by FISH would provide a high degree of clarity that the psDNA is not acting like a tattoo that outlives the intact protein. The FISH method could take advantage of any amplification step as long as it is consistent with detection by scRNAseq. Microscopy could demonstrate that the two signals remain in the same comparments. A particularly powerful way to show the direct association would be to perform a bulk IP-seq with anti-Ova and detection of the bar code with a test of the efficiency of depletion of the bar code form the cell lysate. A well-controlled experiment could be performed to map out the time dependent loss of protein (IP-western or capture immunoassay), and the free and protein associated psDNA. It would be ideal to include a macrophage in the analysis as a highly degradative cells in comparison to the dendritic cell and LEC, that maybe more specialized to regain intact proteins.
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