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closeReferee comments: Referee 3
Posted by PLOS_ONE_Group on 03 Mar 2008 at 12:27 GMT
Referee 3's review:
Review of the original submission of the manuscript
Tetramers are very useful tools for studying T cell responses in vivo. They are sometimes difficult and nearly always timeconsuming to make, however. In this MS the authors describe a rapid method for production of tetramers that include MHC class I proteins + peptides. The method should save investigators a fair amount of time and money if it works.
The major concenr with this MS is not specific to the authors' tetramers but applies also to the conventionally made reagents used as controls. The problem shows up in Figures 4 and 6 in which the staining with the MHC/peptide oligomers versus CD8 is shown. In all cases there is a very pronounced diagonal of the plot of the two stainings versus each other. This usually means that the two reagents are to some extent staining the same molecule. Class I tetramers sometimes have an affinity for CD8 (since the two bind each other) and this binding is unrelated to the MHC/peptide specificity of the tetramer. Why do the authots see this dioagonal staining in tetramer versus CD8 staining plots? Do they worry about nonspecific binding of the MHC oligomers to CD8? How do they know this is not occuring?
The authors must resolve this issue before the MS can be published.
Review of the first revised manuscript
The authors have dalt with my comment and the MS is now suitable for publication in PLOSone with the on line addition of the explanation for the diagonal that the authors give in their rebuttal.
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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.
RE: Referee comments: Referee 3
SorenBuus replied to PLOS_ONE_Group on 17 Mar 2008 at 14:40 GMT
Answer (specificity in general): Yes, we do worry a lot about specificity. Fig 7 demonstrates the very specific nature of our tetramer interactions: the A2/LMP125 stains T cells of donor 2 and no other donors T cells, the A2/LMP365 tetramer stains T cells of the other donors, but not donor 2 T cells, and A2/LMP416 stains T cells of donor 1 and 2, but not donor 3 and 4 T cells. Thus, the tetramers are all of staining quality, and all T cells are of stainable quality. This is further elaborated on in figure 8, which also demonstrate differential staining of donor T cells, which cannot be explained by non-specific CD8 interaction. These experiments are powerful rebuttals of the issue of CD8 driving non-specific binding.
Answer (diagonal issue): We have reanalyzed our tetramers staining. Using the forward-side scatter plot one can sub-divide the living cells into larger and more granulated cells (blasts) and smaller and less granulated cells (non-blasted cells). If these are projected onto the CD8 vs. tetramer scatter plot it becomes apparent that the smaller cells represent a well-demarcated group of stained cells, and the blasts represent another well-demarcated group of stained cells, within the CD8 vs tetramer plot. Both of these subpopulations are non-diagonal, and it is only the positions of the two subpopulations relative to one another that generate an apparent diagonal. Thus, at the time of analysis the restimulated cells contain a mixture of blasted and non-blasted cells. The blasted cells are brighter staining for both CD8 and TcR than the non-blasted cells. Overall, our experiments do not support the concern that the direct interaction of HLA with CD8 affects the enumeration of tetramer+ T cells.