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2015-Wong et al-Cell Reports

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mleipold

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Post Mon Jun 22, 2015 3:21 pm

2015-Wong et al-Cell Reports

"Mapping the Diversity of Follicular Helper T Cells in Human Blood and Tonsils Using High-Dimensional Mass Cytometry Analysis"
Wong Michael T; Chen Jinmiao; Narayanan Sriram; Lin Wenyu; De Lafaille Maria Alicia Curotto; Poidinger Michael; Anicete Rosslyn; Kiaang Henry Tan Kun; Newell Evan W
Cell Reports, 2015
http://dx.doi.org/10.1016/j.celrep.2015.05.022
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mkunicki

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Post Mon Jun 22, 2015 5:39 pm

Re: 2015-Wong et al-Cell Reports

Hello,

This question is for anyone using CyTOF to evaluate CD4 regulatory or helper T cells.


I have consistently found (both personally and in publication) that the viSNE map for this cell population merges the CD4 subpopulations, which could be a testament to the natural heterogeneity within CD4 T cells. This article and it's reference (Becher, et al. Nature. Oct 2014.) to me use the best method of unbiassedly selecting potential CD4 subpopulations (DensVM) for viSNE maps. Does anyone here have any better suggestions for automated gating strategies after viSNE processing? This step seems to be the crux in studying this cell type on CyTOF.

As an aside, I have a hard time believing this publication does not have a significant level of background in many of their channels when looking at their supplementary information. Is this not true, or likely not an issue for the authors?


Best,
Matthew
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mleipold

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Post Mon Jun 22, 2015 11:04 pm

Re: 2015-Wong et al-Cell Reports

Hi Matthew,

Would you clarify what you mean by "significant level of background in many of their channels when looking at their supplementary information"? I just want to make sure that I understand what you're meaning.

If you can give specific examples from the Supplementary Info, that would be helpful.


Mike
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Evan

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Post Tue Jun 23, 2015 6:27 am

Re: 2015-Wong et al-Cell Reports

Hi Matthew,

Thanks for your comment. For both of these papers, our goal was to better understand the composition of the cell populations. I agree that tSNE does a very good job at delineating sub-populations in a visually verifiable way, even when some are quite rare. Nonetheless, some populations of interest such as Th2 or other rare helper subsets do not always nicely segregate. This is probably because they are too rare and/or because there aren’t enough other defining features (e.g., consistent expression or other markers being used – or they are also heterogeneous, etc..). Furthermore, as you point out, there are tradeoffs in terms of optimizing signal for all the markers we are using. So, for some markers, we still have some room for improvement. We included examples of each of the stains within this dataset in the supplementary material to illustrate these limitations. Although better staining may help to improve the tSNE plots and the resolution of these populations, we are satisfied with our ability to set objective and mostly accurate thresholds for defining cells that are positive (and high/low in some cases) vs. negative for each of the markers based on these plots and on quite clean plots of unstimulated cells we ran using the same panel.

In terms of clustering algorithms, we agree with your comment and used densVM because it performed better (visually) than other methods we compared. However, I think that there is room for improvement here as well in that some small but visually discernible clusters are not always identified and other large amorphous clusters seem to be arbitrarily segregated. I think that continued evaluation and improvement of clustering algorithms would be useful in this respect. I also think that manually defined cluster boundaries (taking advantage of the visual nature of dimensionality reduction methods!) are also useful and usually more accurate at this stage. That is, if using subjective gates can be accommodated in the analysis, I think it makes good sense to just gate on them by hand. I’d argue that clustering algorithms and choices of parameters for these algorithms are always going to be subjective anyway - but of course it depends on the purpose.

Cheers!

Evan and Mike W.

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