Peer review of: Mesenchymal stem cells offer a drug-tolerant and immune-privileged niche to (2019)


  

 

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Major comment: The majority of comments have been well addressed by the authors. The additional 
work and explanations are appreciated. 
 
Remaining comments: The immunohistochemistry dual stain (red and brown) is much improved but 
not quite convincing on the computer screen. There are 2 reasons: First - the magnification may be 
too low, and the authors could consider increasing the magnification to 400x dry objective, or 1000x 
with oil immersion? Second - negative controls (tissue sections incubated with isotype-matched 
antibody) seem to be lacking. Negative controls would help determine the non-specific staining. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
My original comment: ” Fig4 I: In contrast to Fig4H, Fig4I is not convincing. First larger orange arrows 
lie on top of smaller arrows, if you zoom into the picture. This is misleading and should be avoided. 
Irrespective of the arrows the CD73 positive cells cannot be identified with the blue counterstaining 
within the pictures of the tissue sections provided. This needs to readdressed in detail.” 
 
Author reply: “We have replaced figures 4H and 4I with a much higher resolution images showing 
clear distinction between the dual staining. The arrows are shown prominently and we believe the 
histopathology data in the revised manuscript matches up to the desired standard.” 
 
• The authors have replaced figures 4H and 4I with novel IHC images with higher resolution. The 
arrows are now clearly visible. However the histopathology data do not meet the expected standards 
(s. below). 
 
My original comment: “The authors do state in their text that Ag85 and CD73 are “in close vicinity” 
but this is in fact several cell diameters away. It appears that in contrast to their Bl6 mouse data, the 
authors do not see CD73 cells harboring Mtb bacteria in human TB lesions. The apparent key question 
is: Do MSC in the human TB patient harbor Mtb bacteria as the mouse data suggest, or does that 
differ. In order to address this the authors may consider e.g. a version of a proximity ligation assays 
(e.g. CD73 vs Ag85) to demonstrate the presence of Mtb within MSC or may use an independent 
staining protocol, where the signals can be undoubtedly visualized and clearly separated from each 
other. Another way to address this may be the use of a Ziehl-Neelsen stain followed by IHC for CD73.” 
 
Author reply: Previously, the dual staining in human granuloma samples were not as well standardized 
and therefore we could not really see their co-localization. In the revised manuscript we indeed show 
clear co-localization between CD73 and Mtb. In addition, we also observe similar co-localization 
between CD105 (another MSC marker) and Mtb. Therefore we have firmed up our inference here and 
instead of saying “in close vicinity”, we do mention “MSC harboring Mtb could be seen in and around 
the granulomas”. 
 
• The overall quality with regard to the provided IHC data has improved. However, the authors did not 
appropriately address the question whether MSC in the human TB patient harbor Mtb bacteria as the 
mouse data suggest. The presented stainings are not convincing. I disagree with the authors that the 
provided data allow that the statement that “MSC harboring Mtb could be seen in and around the 
granulomas”. 

 
o The use of a red (CD73 and CD105) and brown (MtbAg85B) detection system within one tissue 
section does not allow the the identification of double positive cells in the provided data sets. The 
independent signals cannot be undoubtedly visualized and therefore cannot be clearly separated from 
each other. 
o In contrast to the very clear and prominent detection of CD73 and CD105 by IHC, the Ag85B 
stainings are of poor quality. The brown staining for Ag85B is very weak. Prominently stained Ag85B-
positive cells cannot be detected in the provided figures, thus double positive cells cannot be 
identified. The black arrows do not clearly identify Ag85B-positive cells. 
o The authors should either provide additional immunofluorescence (IF) double stainings for 
CD73/Ag85B and CD105/Ag85B or perform a Ziehl-Neelsen stain followed by IHC for CD73 or CD105 
as suggested previously.