Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens

Reviewer #1 (Public Review):

Summary:
The authors develop a method to fluorescently tag peptides loaded onto dendritic cells using a two-step method with a tetracystein motif modified peptide and labelling step done on the surface of live DC using a dye with high affinity for the added motif. The results are convincing in demonstrating in vitro and in vivo T cell activation and efficient label transfer to specific T cells in vivo. The label transfer technique will be useful to identify T cells that have recognised a DC presenting a specific peptide antigen to allow the isolation of the T cell and cloning of its TCR subunits, for example. It may also be useful as a general assay for in vitro or in vivo T-DC communication that can allow the detection of genetic or chemical modulators.

Strengths:
The study includes both in vitro and in vivo analysis including flow cytometry and two-photon laser scanning microscopy. The results are convincing and the level of T cell labelling with the fluorescent pMHC is surprisingly robust and suggests that the approach is potentially revealing something about fundamental mechanisms beyond the state of the art.

Weaknesses:
The method is demonstrated only at high pMHC density and it is not clear if it can operate at at lower peptide doses where T cells normally operate. However, this doesn't limit the utility of the method for applications where the peptide of interest is known. It's not clear to me how it could be used to de-orphan known TCR and this should be explained if they want to claim this as an application. Previous methods based on biotin-streptavidin and phycoerythrin had single pMHC sensitivity, but there were limitations to the PE-based probe so the use of organic dyes could offer advantages.

Reviewer #2 (Public Review):

Summary:
The authors here develop a novel Ovalbumin model peptide that can be labeled with a site-specific FlAsH dye to track agonist peptides both in vitro and in vivo. The utility of this tool could allow better tracking of activated polyclonal T cells particularly in novel systems. The authors have provided solid evidence that peptides are functional, capable of activating OTII T cells, and that these peptides can undergo trogocytosis by cognate T cells only.

Strengths:
-An array of in vitro and in vivo studies are used to assess peptide functionality.
-Nice use of cutting-edge intravital imaging.
-Internal controls such as non-cogate T cells to improve the robustness of the results (such as Fig 5A-D).
-One of the strengths is the direct labeling of the peptide and the potential utility in other systems.

Weaknesses:
1. What is the background signal from FlAsH?
The baselines for Figure 1 flow plots are all quite different. Hard to follow. What does the background signal look like without FLASH (how much fluorescence shift is unlabeled cells to No antigen+FLASH?). How much of the FlAsH in cells is actually conjugated to the peptide? In Figure 2E, it doesn't look like it's very specific to pMHC complexes. Maybe you could double-stain with Ab for MHCII. Figure 4e suggests there is no background without MHCII but I'm not fully convinced. Potentially some MassSpec for FLASH-containing peptides.

2. On the flip side, how much of the variant peptides are getting conjugated in cells? I'd like to see some quantification (HPLC or MassSpec). If it's ~10% of peptides that get labeled, this could explain the low shifts in fluorescence and the similar T cell activation to native peptides if FlasH has any deleterious effects on TCR recognition. But if it's a high rate of labeling, then it adds confidence to this system.

3. Conceptually, what is the value of labeling peptides after loading with DCs? Why not preconjugate peptides with dye, before loading, so you have a cleaner, potentially higher fluorescence signal? If there is a potential utility, I do not see it being well exploited in this paper. There are some hints in the discussion of additional use cases, but it was not clear exactly how they would work. One mention was that the dye could be added in real-time in vivo to label complexes, but I believe this was not done here. Is that feasible to show?

4. Figure 5D-F the imaging data isn't fully convincing. For example, in 5F and 2G, the speeds for T cells with no Ag should be much higher (10-15micron/min or 0.16-0.25micron/sec). The fact that yours are much lower speeds suggests technical or biological issues, that might need to be acknowledged or use other readouts like the flow cytometry.