Studying the biology of cytotoxic T lymphocytes in vivo with a fluorescent granzyme B-mTFP knock-in mouse

Acceptance summary:

This manuscript describes a new and interesting mouse model to study CTL (and probably NK cell) activity in vivo by tracking Granzyme B levels and localisation in real-time. This will be an excellent resource for groups studying a range of T and NK cell functions in many contexts from anti-viral defences to tumour immunotherapy.

Decision letter after peer review:

Thank you for submitting your article "Studying the biology of cytotoxic T lymphocytes in vivo with a fluorescent Granzyme B-mTFP knock-in mouse" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Michael L Dustin as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Satyajit Rath as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Jennifer L Stow (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

Advanced in vivo microscopy offers the ability to track fluorescently tagged cells, organelles and proteins in their native tissue environments providing invaluable insights for physiology and pathophysiology. Moreover, the ability to do so seamlessly, without perturbing endogenous localization or function, is the ultimate goal for such studies. The behaviour of cytotoxic granules (CGs) in T cells has long been a preoccupation of immunologists and others, due to their essential role in cell-cell killing, now a topic of intense interest in cancer immunotherapies.

Your study describes the construction of a fluorescent fusion protein GzmB- mTFP consisting of mTFP, a cleavable linker and the CG enzyme, granzymeB and the generation of a knock-in mouse expressing this probe in CTLs. Elegant imaging is used to show remarkable localization and colocalization of the fusion protein and other markers in CGs. The behaviour of the fusion protein and CGs are carefully measured in ex vivo cells and in mice, to ensure no aberrant features have been introduced. With no impediment to normal function, this CG probe appears to be superior to others available. Finally, the WT and KI mice are subjected to allografts in the eye to provide a very nice demonstration in vivo of cell infiltration and granule formation in CTLs. More sophisticated studies in the future no doubt will allow discrimination of new aspects of CG behaviour in cell-cell killing using further super-resolution microscopy and CLEM.

Revisions for this paper:

You are commended on a rigorously performed study, the KI mouse and its protein GzmB-mTFP cells will be extremely valuable for many investigators and the mice have the hallmarks of becoming iconic models for future CG studies. As such, this study represents a major, enabling contribution to the field. There are some issues that should be addressed. First of all, as this will be a resource for the field, how will you make the mice available to the community?

If the data are available some quantification of granzyme and fusion protein levels in Figures 1 C and D would be helpful since the relative levels in WT and KI mice appear to differ between these panels. It would certainly be interesting in the future to follow the expression of the fusion protein over longer time periods and under different stimulation conditions in the KI mice.

What is the expression of the reporter in NK cells and activated CD4 T cells? A more detailed description of the model would be welcome. In the in vivo experiment, it would be important that the author stained for CD8 T cell and NK cells. A negative result on a CD4 staining is not enough to claim that cells are CD8 T cells. For the in vivo imaging in general it would be important to show controls with T1D and similar/identical imaging conditions in the micro without the knockin. The green fluorescence channel has a lot of autofluorescence and that autofluorescence might appear granular, so it would be helpful to see evidence that this is specific mTFP fluorescence.

Revisions expected in follow-up work:

Do you have a way to look at the kinetics of the cleavage process, perhaps through a pulse-chase experiment or cruder kinetic study of granule formation during CTL maturation.

In this context, could the authors comment on whether they see any evidence for cleavage of the fusion protein before the CG (eg at the level of post-Golgi vesicles) where proteolytic processing is possible? Can cleavage be prevented in the lysosome/granules after dissipating the pH drop, or by incubation of the cells with protease inhibitors that would intersect at the endo-lyosomal comparments? This is not necessary to do immediately but of future interest, but if you have data on this it would strengthen it.

Can you draw new conclusions from the longitudinal analyse of the T1D model? New biology is not needed at this stage, but it should be clear that the tools will be useful for generating quantitative data to address open questions in biology.