Reviewer ’s report  
 
Title: WNT5A Transforms Intestinal CD8αα+ IELs into an Unconventional Phenotype with Pro -
inflammatory Features  
 
Version: 0 Date: 13 Sep 2015  
 
Reviewer: Govind Bhagat  
 
Reviewer ’s report:  
 
This report is intriguing and provides interesting data on the role of WNT pathway acti vation on 
colonic IELs. The language needs considerable editing for language. My specific comments are 
listed below:  
 
1. The methods for IEL isolation should be better described. Did the authors find similar 
yields in the small intestine as the colon? The total yield of IELs in the mouse colon 
samples needs to be described. Was the same site and amount of colon specimen used for 
IEL extraction? What was the range and median of IELs in normal and inflamed colonic 
tissue? Was this within range of what has bee n published? These data will be useful to 
mucosal immunologists.  
 
2. Please describe the phenotype and percentage of IEL subsets obtained by the authors. 
We're there any a changes between the normal and inflamed mouse colon's with regards 
to percentage of T CR alpha -beta, TCR gamma -delta, CD8, CD4 etc.  
 
3. Did the authors also study the response of other IEL subsets for WNT family member 
expression and response to inhibitors? I would recommend to do so, as these would serve 
as additional controls and also provide information about the restricted or genera l WNT 
pathway activation (canonical and non -canonical). Was there any difference between the 
CD8 alpha -alpha TCR alpha -beta and TCR gamma -delta IELs with regard to WNT 
pathway activation in inflamed mouse colon?  
 
4. Why did the authors compare the data rega rding methylation profiles of whole inflamed 
human ulcerative colitis samples with mouse IELs. As the authors report, the signal to 
noise ratio was inadequate to study WNT activation when they used whole mouse 
(inflamed) colon compared to the IEL fraction.  In my opinion, the human tissue analysis 
does not prove or disprove the authors results. Due to the substantial lamina propria 
inflammation in ulcerative colitis, the authors findings likely reflect the WNT activation 
status in this compartment rather tha n the IELs. It is difficult to obtain enough IELs from 
human colonic samples, however, the authors could try to obtain fresh biopsies and 
evaluate the canonical and non -canonical WNT pathway activation by a targeted PCR 
approach on isolated IELs and compar e them with normal 'control' human colon biopsy 
samples. Alternatively, if suitable antibodies exist to evaluate canonical and non -
canonical WNT activation in formalin -fixed, paraffin -embedded human IBD colon 
biopsy samples, these data would also be suppor tive of the authors findings in the mouse 
colon. If these studies cannot be performed then I would recommend removing the 
human correlative studies as they are meaningless due to the aforementioned reasons.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are  required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of written English  
Please indicate the quality o f language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received  reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gai n or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salar y from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answe r no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand t hat my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be mad e available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, whi ch will not be published  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: WNT5A Transforms Intestinal CD8αα+ IELs into an Unconventional Phenotype with Pro -
inflammatory Features  
 
Version: 0 Date: 13 Sep 2015  
 
Reviewer: Govind Bhagat  
 
Reviewer ’s report:  
 
This report is intriguing and provides interesting data on the role of WNT pathway acti vation on 
colonic IELs. The language needs considerable editing for language. My specific comments are 
listed below:  
 
1. The methods for IEL isolation should be better described. Did the authors find similar 
yields in the small intestine as the colon? The total yield of IELs in the mouse colon 
samples needs to be described. Was the same site and amount of colon specimen used for 
IEL extraction? What was the range and median of IELs in normal and inflamed colonic 
tissue? Was this within range of what has bee n published? These data will be useful to 
mucosal immunologists.  
 
2. Please describe the phenotype and percentage of IEL subsets obtained by the authors. 
We're there any a changes between the normal and inflamed mouse colon's with regards 
to percentage of T CR alpha -beta, TCR gamma -delta, CD8, CD4 etc.  
 
3. Did the authors also study the response of other IEL subsets for WNT family member 
expression and response to inhibitors? I would recommend to do so, as these would serve 
as additional controls and also provide information about the restricted or genera l WNT 
pathway activation (canonical and non -canonical). Was there any difference between the 
CD8 alpha -alpha TCR alpha -beta and TCR gamma -delta IELs with regard to WNT 
pathway activation in inflamed mouse colon?  
 
4. Why did the authors compare the data rega rding methylation profiles of whole inflamed 
human ulcerative colitis samples with mouse IELs. As the authors report, the signal to 
noise ratio was inadequate to study WNT activation when they used whole mouse 
(inflamed) colon compared to the IEL fraction.  In my opinion, the human tissue analysis 
does not prove or disprove the authors results. Due to the substantial lamina propria 
inflammation in ulcerative colitis, the authors findings likely reflect the WNT activation 
status in this compartment rather tha n the IELs. It is difficult to obtain enough IELs from 
human colonic samples, however, the authors could try to obtain fresh biopsies and 
evaluate the canonical and non -canonical WNT pathway activation by a targeted PCR 
approach on isolated IELs and compar e them with normal 'control' human colon biopsy 
samples. Alternatively, if suitable antibodies exist to evaluate canonical and non -
canonical WNT activation in formalin -fixed, paraffin -embedded human IBD colon 
biopsy samples, these data would also be suppor tive of the authors findings in the mouse 
colon. If these studies cannot be performed then I would recommend removing the 
human correlative studies as they are meaningless due to the aforementioned reasons.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are  required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of written English  
Please indicate the quality o f language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received  reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gai n or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salar y from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answe r no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand t hat my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be mad e available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, whi ch will not be published  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: WNT5A Transforms Intestinal CD8αα+ IELs into an Unconventional Phenotype with Pro -
inflammatory Features  
 
Version: 0 Date: 13 Sep 2015  
 
Reviewer: Govind Bhagat  
 
Reviewer ’s report:  
 
This report is intriguing and provides interesting data on the role of WNT pathway acti vation on 
colonic IELs. The language needs considerable editing for language. My specific comments are 
listed below:  
 
1. The methods for IEL isolation should be better described. Did the authors find similar 
yields in the small intestine as the colon? The total yield of IELs in the mouse colon 
samples needs to be described. Was the same site and amount of colon specimen used for 
IEL extraction? What was the range and median of IELs in normal and inflamed colonic 
tissue? Was this within range of what has bee n published? These data will be useful to 
mucosal immunologists.  
 
2. Please describe the phenotype and percentage of IEL subsets obtained by the authors. 
We're there any a changes between the normal and inflamed mouse colon's with regards 
to percentage of T CR alpha -beta, TCR gamma -delta, CD8, CD4 etc.  
 
3. Did the authors also study the response of other IEL subsets for WNT family member 
expression and response to inhibitors? I would recommend to do so, as these would serve 
as additional controls and also provide information about the restricted or genera l WNT 
pathway activation (canonical and non -canonical). Was there any difference between the 
CD8 alpha -alpha TCR alpha -beta and TCR gamma -delta IELs with regard to WNT 
pathway activation in inflamed mouse colon?  
 
4. Why did the authors compare the data rega rding methylation profiles of whole inflamed 
human ulcerative colitis samples with mouse IELs. As the authors report, the signal to 
noise ratio was inadequate to study WNT activation when they used whole mouse 
(inflamed) colon compared to the IEL fraction.  In my opinion, the human tissue analysis 
does not prove or disprove the authors results. Due to the substantial lamina propria 
inflammation in ulcerative colitis, the authors findings likely reflect the WNT activation 
status in this compartment rather tha n the IELs. It is difficult to obtain enough IELs from 
human colonic samples, however, the authors could try to obtain fresh biopsies and 
evaluate the canonical and non -canonical WNT pathway activation by a targeted PCR 
approach on isolated IELs and compar e them with normal 'control' human colon biopsy 
samples. Alternatively, if suitable antibodies exist to evaluate canonical and non -
canonical WNT activation in formalin -fixed, paraffin -embedded human IBD colon 
biopsy samples, these data would also be suppor tive of the authors findings in the mouse 
colon. If these studies cannot be performed then I would recommend removing the 
human correlative studies as they are meaningless due to the aforementioned reasons.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are  required in your comments to the authors.  
 
No 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript or would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
Not relevant to this manuscript  
 
Quality of written English  
Please indicate the quality o f language in the manuscript:  
 
Not suitable for publication unless extensively edited  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received  reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gai n or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salar y from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
 
If you can answe r no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer review policy of the journal. I understand t hat my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be mad e available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as confidential comments  
to the editors, whi ch will not be published  
 
I agree to the open peer review policy of the journal  
assistant