Download OX40 ligand newly expressed on bronchiolar progenitors mediates

EMBO Molecular Medicine
Peer Review Process File - EMM-2015-06154
OX40 ligand newly expressed on bronchiolar progenitors
mediates influenza infection and further exacerbates
pneumonia
Taizou Hirano, Toshiaki Kikuchi, Naoki Tode, Arif Santoso, Mitsuhiro Yamada, Yoshiya
Mitsuhashi, Riyo Komatsu, Takeshi Kawabe, Takeshi Tanimoto, Naoto Ishii, Yuetsu Tanaka,
Hidekazu Nishimura, Toshihiro Nukiwa, Akira Watanabe, and Masakazu Ichinose
Corresponding author: Toshiaki Kikuchi, Niigata University Graduate School of Medical and
Dental Sciences
Review timeline:
Submission date:
Editorial Decision:
Resubmission:
Editorial Decision:
Revision received:
Accepted:
15 January 2015
01 February 2015
16 December 2015
11 January 2016
11 February 2016
16 February 2016
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
Editor: Céline Carret
1st Editorial Decision
01 February 2015
Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now
heard back from the two referees whom we asked to evaluate your manuscript. Although the
referees find the study to be of potential interest, they also raise a number of concerns that must be
addressed in the next final version of your article.
As you will see from the comments below, both referees find the study importing and novel.
However, they both recommend performing additional experiments and replicating some of the key
data to improve and strengthen the findings. As the reports are very clear and explicit I will not get
into further details, but please note that it is EMBO Molecular Medicine policy to allow only a
single round of revision and that, as acceptance or rejection of the manuscript will depend on
another round of review, your responses should be as complete as possible.
Please see instructions below for submitting your revised article. Please do not forget to include the
authors' checklist and make sure that all requested information can be found within the main text.
I look forward to seeing a revised form of your manuscript as soon as possible.
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***** Reviewer's comments *****
Referee #1 (Comments on Novelty/Model System):
Some additional experiments, and repeating some experiments, as specified in the remarks to the
author, should enhance the conclusions in the paper and make it more convincing.
Referee #1 (Remarks):
This is a very interesting paper, suggesting that OX40L can be a receptor for influenza virus and aid
infection of bronchial epithelial cells. In general the experiments are quite convincing but additional
experiments are needed to solidify and enhance the conclusions.
1. In Figure 1a, the authors suggest that OX40L-/- mice are protected from death with high dose
intratracheal PR8/H1N1 infection whereas OX40-/- mice are not. This is perhaps the most important
data as it leads the rest of the paper in supporting the notion that the any results on lung pathology
are not simply a repeat of what has been published before, which claimed that OX40-OX40L
interactions drive an exacerbated T cell response to flu virus that causes pathology (Humphreys et
al, JEM, 2003). Although the data as presented look convincing, this experiment was apparently
only 1 experiment with 12 mice per group, and it is not indicated how survival was measured
(presumably a loss of a certain amount of body weight given animal welfare rules). There are some
differences in BAL counts and histology that somewhat correlate with the survival curves, but these
are not so different to be convincing that this would fully explain the survival results. In Fig. 1d
body weight loss in BM chimeras is shown, with very little change between groups, again
questioning how real the data are in Fig. 1a. Thus, the contention that OX40-/- mice differ from
OX40L-/- mice needs to be substantiated, with more repeat experiments, showing weight loss curves
as well as survival curves. Importantly this should be performed with different doses of the virus to
show the result does not simply apply to one specific situation.
2. The authors suggest that OX40L is induced on bronchiolar progenitors by both HIN1 infection
(Fig. 2H) and H3N2 infection (Fig. 3B) but the latter results are not convincing that OX40L is
significantly or strongly upregulated. Again, as the conclusion of the paper hinges on OX40L being
active on these cells independent of OX40, experiments should also be performed assessing weight
loss and survival in OX40L-/- and OX40-/- with H3N2 to show that the results are not simply
specific for one strain of flu.
3. The authors imply that OX40L-/- mice are protected from lethal flu infection in part because
bronchiolar progenitor cells cannot be infected equivalently due to OX40L directly binding the
virus. They show M2 expression in WT vs OX40L-/- mice (Fig. 4G). If this is significant, and again
not related to an immune response involving OX40-OX40L interactions, a kinetic analysis of levels
of total virus particles (conventional plaque assay) as well as M2 protein in the lungs of OX40L-/and OX40-/- mice over the first week of infection should also be dramatically different. Performing
the experiment with H1N1 and H3N2 would again substantiate the conclusions.
4. There is a disconnect in the relative NP expression in mouse OX40L transfected cells in Fig. 6A
vs 6D. Can the authors explain why the qPCR results are substantially different from the semiqPCR.
5. In Fig. 7, the authors present data with human OX40L transfected cells treated with sialidase. It
would also be useful to perform the same study with mouse OX40L.
6. The authors need to control for OX40L transfection in MDCK cells. Based on data in Fig. 8, it is
not clear how uniform OX40L is expressed and the implication is that only a subset of cells may
have expressed high amounts. This could impact the viral infection data. Also the data with the
mutant OX40L constructs can only be evaluated effectively if similar levels of OX40L are
expressed on the cell surface with each mutant. What are the absolute levels of OX40L on
transfected cells and how do they compare to bronchiolar progenitors.
7. The final data staining OX40L in tissue sections from autopsy are not convincing. The data are
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not clear and it is impossible to see any real staining. Moreover, bronchial epithelial cells are
notoriously known to stain with almost any antibody. There is no control for the antibody to show it
stains anything specifically. The comparison between tissues is also not valid, and making
conclusions from tissues from single patients whose histories are not documented also calls into
question any conclusions drawn.
Minor point:
Some of the data are not logically placed. e.g. the first data on sialic acids (Fig. 4H and I) would be
better in the current Fig. 6. There is no need for a separate Fig. 5 as this data is related to that in Fig.
4. Similarly, data in Fig. 3C and D are not mentioned until Fig. 7 is discussed and would be better in
Fig. 7. Also, data in Fig. 8 should either be in a supplementary Figure, or data in Fig. 8 combined
with Fig. 7G-J in the final Figure.
Referee #2 (Comments on Novelty/Model System):
The mouse model is adequate to study influenza A virus infection. The authors use mainly PR8
virus, which is highly mouse adapted and used in many laboratories in mice.
Referee #2 (Remarks):
The authors show that OX40L-deficient mice are more resistant to challenge with PR8 virus
compared to wild type mice. This correlates with reduced survival and cellular infiltration in the
lungs. Bone marrow transplantation experiments provide some evidence that loss of OX40L
expression on stromal cells is responsible for the increased resistance to influenza virus infection,
although reduced cellular infiltration in OX40L-/- mice is not observed in these experiments. M2staining of lung sections is reduced in OX40L knockout mice. Influenza virus infection leads to an
increase of the number of bronchiolar progenitor cells and a decrease in the number of club cells.
Ox40L mRNA levels are upregulated in BP cells following influenza virus infection. Similar
findings are shown for an H3N2 virus infection. Infection of Ox40L negative mice is associated
with reduced levels of NP mRNA in ex vivo isolated cells and reduced M2 expression. BP and Club
cells both bind SN lectin, suggesting the presence of alpha 2-6 linked sialic acid residues on their
cell surface. Transfection of MDCK cells with an Ox40L expression vector is associated with
increased production of NP mRNA and M2 expression after influenza virus infection. Conversely, a
mAb directed against Ox40L reduces NP expression and partially protects mice against PR8
challenge. Site specific mutagenesis experiments and transfection of MDCK cells suggest that the
N-glycosylation site at position 90 is important for Ox40L-dependent increased influenza gene
expression. Immuno-histological stains of lung section from an influenza and a bacterial pneumonia
victim are shown in an attempt to document the clinical relevance of Ox40L expression for the
outcome of influenza virus infection in human.
The main message of the paper is that Ox40 ligand, which is type II membrane protein that has
important T cell co-stimulatory functions, serves as a receptor on bronchial epithelial progenitor
cells for influenza A virus infection. Loss of Ox40L, blocking Ox40L with mAb or removing certain
N-glycans on Ox40L would then lead to reduced influenza A virus burden and associated disease.
This is novel and interesting concept. However, the authors seem to ignore the role of Ox40L in
immune cell stimulation as a possible explanation for the increased survival in the knock out mice.
In addition, their findings are not in line with published data on the lack of a phenotype for influenza
in mice that lack alpha 2,6 sialyltransferase. Finally, at no point is live virus replication determined.
I recommend that the authors perform extra experiments to strengthen their case.
Major remarks
1. The authors rely on RT-PCR for NP and on immunostaining of M2 to document "viral burden".
The qRT-PCR data should be improved because they rely on a single house keeping gene for
normalization. I recommend the authors to seek advice for improving the qRT-PCR data in
Derveaux et al, Methods, 2010. In addition, the authors should determine newly produced virus
from infected mice (wt and Ox40L-/- and MDCK cells transfected with Ox40L or empty vector) by
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plaque assay or by quantifying cytopathic effects in susceptible cells. MDCK cells are typically used
for this.
2. Ox40L is a co-stimulatory molecule for T cells. An explanation for the increased survival of
Ox40L-/- mice could be a reduced level of pro-inflammatory cytokines. In fact, the reduced cellular
infiltration in the BAL (Fig 1B) suggests that this may be implicated. The authors should compare
the amounts of a number of cytokines such as IL4, IL6 and IFN gamma in BAL after infection in wt
and knock out mice.
3. The findings are at odds with the paper by Glaser et al (Virus Res. 2007) that shows no significant
impact of loss of alpha 2,6 sialic acid on N-glycans on susceptibility of mice to human influenza A
virus infection. This should be discussed by the authors.
4. Why is there no more difference in cell infiltration in the radiation chimeras (Fig 1E)?
5. Panels in Fig 4D and 5 reveal very little. Please improve.
6. The authors conclude that club cells are poorly infected by influenza (Fig 4A-E). However, it was
recently reported that these cells do become infected with influenza virus (Heaton et al JEM, 2014).
Please refer to this paper and comment on it.
7. Fig 7: MDCK cells are highly susceptible to influenza virus infection. Why is it that the pNull
control transfected cells do not produce virus? This is unexpected and may be due to the induction
of type I IFN by the transfection procedure. Please determine IFN levels. Reduced expression of e.g.
the Asn90 mutant could equally explain the reduced infectibility of the cells rather than the lack of a
N-glycosylation at position 90 (Fig 7G).
8. The immunostains of the clinical samples in Fig 7 have a very poor resolution. The Ox40L
staining should be performed with a negative control antibody and ideally an influenza A virus
antigen (NP should work) antibody. Micrographs should be prepared with a much better resolution.
9. Please explicitly indicate whether littermates were used or not in the comparison of wt and ko
mice. This is very important because the (gut) microbiota and microbiome composition is known to
affect susceptibility to influenza virus infection.
Minor remarks:
It is hard to follow the figure numbering in the text. E.g. Fig 3D appears at the end of the results
section. Please use ascending figure numbering in the text.
Resubmission
16 December 2015
Referee 1
Referee: (Comments on Novelty/Model System) “Some additional experiments, and repeating some
experiments, as specified in the remarks to the author, should enhance the conclusions in the paper
and make it more convincing.”
Response: We appreciate the Referee’s comments. According to the specified remarks, we have
performed additional experiments.
Referee: (Remarks) “This is a very interesting paper, suggesting that OX40L can be a receptor for
influenza virus and aid infection of bronchial epithelial cells. In general the experiments are quite
convincing but additional experiments are needed to solidify and enhance the conclusions.”
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Response: We appreciate the Referee’s comments.
Referee: “1. In Figure 1a, the authors suggest that OX40L-/- mice are protected from death with
high dose intratracheal PR8/H1N1 infection whereas OX40-/- mice are not. This is perhaps the most
important data as it leads the rest of the paper in supporting the notion that the any results on lung
pathology are not simply a repeat of what has been published before, which claimed that OX40OX40L interactions drive an exacerbated T cell response to flu virus that causes pathology
(Humphreys et al, JEM, 2003). Although the data as presented look convincing, this experiment was
apparently only 1 experiment with 12 mice per group, and it is not indicated how survival was
measured (presumably a loss of a certain amount of body weight given animal welfare rules). There
are some differences in BAL counts and histology that somewhat correlate with the survival curves,
but these are not so different to be convincing that this would fully explain the survival results. In
Fig. 1d body weight loss in BM chimeras is shown, with very little change between groups, again
questioning how real the data are in Fig. 1a. Thus, the contention that OX40-/- mice differ from
OX40L-/- mice needs to be substantiated, with more repeat experiments, showing weight loss curves
as well as survival curves. Importantly this should be performed with different doses of the virus to
show the result does not simply apply to one specific situation.”
Response: We agree with this comment. We have repeated the experiments with different doses of
the virus. These data are shown in the revised manuscript (page 5, paragraph 1, lines 5-7, Figure
S3).
Referee: “2. The authors suggest that OX40L is induced on bronchiolar progenitors by both HIN1
infection (Fig. 2H) and H3N2 infection (Fig. 3B) but the latter results are not convincing that
OX40L is significantly or strongly upregulated. Again, as the conclusion of the paper hinges on
OX40L being active on these cells independent of OX40, experiments should also be performed
assessing weight loss and survival in OX40L-/- and OX40-/- with H3N2 to show that the results are
not simply specific for one strain of flu.”
Response: As suggested, we have included the relevant data of H3N2 in the revised manuscript
(page 5, paragraph 1, lines 5-7, Figure S2).
Referee: “3. The authors imply that OX40L-/- mice are protected from lethal flu infection in part
because bronchiolar progenitor cells cannot be infected equivalently due to OX40L directly binding
the virus. They show M2 expression in WT vs OX40L-/- mice (Fig. 4G). If this is significant, and
again not related to an immune response involving OX40-OX40L interactions, a kinetic analysis of
levels of total virus particles (conventional plaque assay) as well as M2 protein in the lungs of
OX40L-/- and OX40-/- mice over the first week of infection should also be dramatically different.
Performing the experiment with H1N1 and H3N2 would again substantiate the conclusions.”
Response: We agree with the Referee’s comment. We have added data of conventional plaque assay
in the revised manuscript (page 9, paragraph 1, lines 8-10, Figure S6).
Referee: “4. There is a disconnect in the relative NP expression in mouse OX40L transfected cells
in Fig. 6A vs 6D. Can the authors explain why the qPCR results are substantially different from the
semi-qPCR.”
Response: The Referee’s question is valid. In the original manuscript, the expression levels of the
influenza virus NP gene were shown as relative to pNull-transfected cells of Fig. 6A (Fig. 4C in the
revised manuscript) and control antibody-pretreated cells of Fig. 6D (Fig. 4F in the revised
manuscript). This confusing notation could have raised the Referee’s question, because OX40Ltransfected cells of Fig. 6A and control antibody-pretreated cells of Fig. 6D should have the similar
levels. We have redrawn Fig. 6A and shown the expression level as relative to OX40L-transfected
cells (revised manuscript, Figure 4C).
Referee: “5. In Fig. 7, the authors present data with human OX40L transfected cells treated with
sialidase. It would also be useful to perform the same study with mouse OX40L.”
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Response: We agree with the Referee’s point. We have performed the additional experiment of
mouse OX40L-transfected cells treated with sialidase (revised manuscript, page 10, paragraph 2,
lines 13-14, Figure S7).
Referee: “6. The authors need to control for OX40L transfection in MDCK cells. Based on data in
Fig. 8, it is not clear how uniform OX40L is expressed and the implication is that only a subset of
cells may have expressed high amounts. This could impact the viral infection data. Also the data
with the mutant OX40L constructs can only be evaluated effectively if similar levels of OX40L are
expressed on the cell surface with each mutant. What are the absolute levels of OX40L on
transfected cells and how do they compare to bronchiolar progenitors.”
Response: We agree with the Referee. In the revised manuscript, we have added data to compare
the expression levels of human OX40L on MDCK cells that were transfected with mutant human
OX40L genes as well as the wild-type one (revised manuscript, page 11, paragraph 3, lines 5-6,
Figure S10).
Referee: “7. The final data staining OX40L in tissue sections from autopsy are not convincing. The
data are not clear and it is impossible to see any real staining. Moreover, bronchial epithelial cells
are notoriously known to stain with almost any antibody. There is no control for the antibody to
show it stains anything specifically. The comparison between tissues is also not valid, and making
conclusions from tissues from single patients whose histories are not documented also calls into
question any conclusions drawn.”
Response: We agree. Since the data staining OX40L in tissue sections from autopsy are not
convincing to draw any conclusions, we have deleted them in the revised manuscript.
Referee: (Minor point) “Some of the data are not logically placed. e.g. the first data on sialic acids
(Fig. 4H and I) would be better in the current Fig. 6. There is no need for a separate Fig. 5 as this
data is related to that in Fig. 4. Similarly, data in Fig. 3C and D are not mentioned until Fig. 7 is
discussed and would be better in Fig. 7. Also, data in Fig. 8 should either be in a supplementary
Figure, or data in Fig. 8 combined with Fig. 7G-J in the final Figure.”
Response: We agree with the Referee. We have assembled Fig. 4H, 4I, and Fig. 6 of the original
manuscript into new Fig. 4 of the revised manuscript. Also we have combined Fig. 5 of the original
manuscript into Fig. 3 of the revised manuscript. To place logically Fig. 3 and Fig. 8 of the original
manuscript, we have moved them to Fig. S4 and Fig. S11, respectively, in the revised manuscript.
Referee 2
Referee: (Comments on Novelty/Model System) “The mouse model is adequate to study influenza A
virus infection. The authors use mainly PR8 virus, which is highly mouse adapted and used in many
laboratories in mice.”
Response: We appreciate the Referee’s comments.
Referee: (Remarks) “The authors show that OX40L-deficient mice are more resistant to challenge
with PR8 virus compared to wild type mice. This correlates with reduced survival and cellular
infiltration in the lungs. Bone marrow transplantation experiments provide some evidence that loss
of OX40L expression on stromal cells is responsible for the increased resistance to influenza virus
infection, although reduced cellular infiltration in OX40L-/- mice is not observed in these
experiments. M2-staining of lung sections is reduced in OX40L knockout mice. Influenza virus
infection leads to an increase of the number of bronchiolar progenitor cells and a decrease in the
number of club cells. Ox40L mRNA levels are upregulated in BP cells following influenza virus
infection. Similar findings are shown for an H3N2 virus infection. Infection of Ox40L negative mice
is associated with reduced levels of NP mRNA in ex vivo isolated cells and reduced M2 expression.
BP and Club cells both bind SN lectin, suggesting the presence of alpha 2-6 linked sialic acid
residues on their cell surface. Transfection of MDCK cells with an Ox40L expression vector is
associated with increased production of NP mRNA and M2 expression after influenza virus
infection. Conversely, a mAb directed against Ox40L reduces NP expression and partially protects
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mice against PR8 challenge. Site specific mutagenesis experiments and transfection of MDCK cells
suggest that the N-glycosylation site at position 90 is important for Ox40L-dependent increased
influenza gene expression. Immuno-histological stains of lung section from an influenza and a
bacterial pneumonia victim are shown in an attempt to document the clinical relevance of Ox40L
expression for the outcome of influenza virus infection in human.”
Response: We appreciate the Referee’s comments.
Referee: “The main message of the paper is that Ox40 ligand, which is type II membrane protein
that has important T cell co-stimulatory functions, serves as a receptor on bronchial epithelial
progenitor cells for influenza A virus infection. Loss of Ox40L, blocking Ox40L with mAb or
removing certain N-glycans on Ox40L would then lead to reduced influenza A virus burden and
associated disease. This is novel and interesting concept. However, the authors seem to ignore the
role of Ox40L in immune cell stimulation as a possible explanation for the increased survival in the
knock out mice. In addition, their findings are not in line with published data on the lack of a
phenotype for influenza in mice that lack alpha 2,6 sialyltransferase. Finally, at no point is live virus
replication determined. I recommend that the authors perform extra experiments to strengthen their
case.”
Response: We appreciate the Referee’s comments.
Referee: (Major remarks) “1. The authors rely on RT-PCR for NP and on immunostaining of M2 to
document "viral burden". The qRT-PCR data should be improved because they rely on a single
house keeping gene for normalization. I recommend the authors to seek advice for improving the
qRT-PCR data in Derveaux et al, Methods, 2010.”
Response: We agree with this comment. We have performed the additional experiment using
normalization against 2 more reference genes, and have achieved similar results (revised
manuscript, page 7, paragraph 2, lines 9-11, Figure S5).
Referee: “In addition, the authors should determine newly produced virus from infected mice (wt
and Ox40L-/- and MDCK cells transfected with Ox40L or empty vector) by plaque assay or by
quantifying cytopathic effects in susceptible cells. MDCK cells are typically used for this.”
Response: We agree and have performed the experiments of plaque assay. The data are shown in
the revised manuscript (page 9, paragraph 1, lines 8-10, Figure S6; page 11, paragraph 2, lines 7-11,
Figure S8).
Referee: “2. Ox40L is a co-stimulatory molecule for T cells. An explanation for the increased
survival of Ox40L-/- mice could be a reduced level of pro-inflammatory cytokines. In fact, the
reduced cellular infiltration in the BAL (Fig 1B) suggests that this may be implicated. The authors
should compare the amounts of a number of cytokines such as IL4, IL6 and IFN gamma in BAL after
infection in wt and knock out mice.”
Response: We agree with the Referee. We have evaluated the amounts of cytokines suggested by
the Referee. These data are shown in the revised manuscript (page 4, paragraph 2, line 9-page 5,
paragraph 1, line 5, Figure S1).
Referee: “3. The findings are at odds with the paper by Glaser et al (Virus Res. 2007) that shows no
significant impact of loss of alpha 2,6 sialic acid on N-glycans on susceptibility of mice to human
influenza A virus infection. This should be discussed by the authors.”
Response: As suggested, we discussed this issue in the revised manuscript (page 16, paragraph 1,
lines 5-8).
Referee: “4. Why is there no more difference in cell infiltration in the radiation chimeras (Fig
1E)?”
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Response: This is probably due to irradiation of the recipients and reconstruction of the immune
system in engraftment of bone marrow. This point has been discussed in the revised manuscript
(page 14, paragraph 1, lines 11-14).
Referee: “5. Panels in Fig 4D and 5 reveal very little. Please improve.”
Response: We agree with the Referee, and have improved them in Figure 3D and 3F of the revised
manuscript.
Referee: “6. The authors conclude that club cells are poorly infected by influenza (Fig 4A-E).
However, it was recently reported that these cells do become infected with influenza virus (Heaton
et al JEM, 2014). Please refer to this paper and comment on it.”
Response: We agree with the Referee’s comment. We have referred the paper and commented on it
in the revised manuscript (page 15, paragraph 1, lines 1-5).
Referee: “7. Fig 7: MDCK cells are highly susceptible to influenza virus infection. Why is it that the
pNull control transfected cells do not produce virus? This is unexpected and may be due to the
induction of type I IFN by the transfection procedure. Please determine IFN levels. Reduced
expression of e.g. the Asn90 mutant could equally explain the reduced infectibility of the cells rather
than the lack of a N-glycosylation at position 90 (Fig 7G).”
Response: We agree with the Referee. We have examined the expression levels of IFN-α and IFN-β
in naive MDCK cells as well as human OX40L-, 90Asn→Ala mutant-, and pNull-transfected
MDCK cells, and found no significant differences between naive and transfected cells. The data are
shown in the revised manuscript (page 11, paragraph 2, lines 11-13, Figure S9). Moreover, to
address the concerns about interference of the transfection procedure, we have specified that the
transfected MDCK cells were washed twice before the influenza infection (revised manuscript, page
19, paragraph 1, lines 5-6).
Referee: “8. The immunostains of the clinical samples in Fig 7 have a very poor resolution. The
Ox40L staining should be performed with a negative control antibody and ideally an influenza A
virus antigen (NP should work) antibody. Micrographs should be prepared with a much better
resolution.”
Response: According to the relevant comment of Referee 1, we have deleted the immunostaining of
the clinical samples in the revised manuscript.
Referee: “9. Please explicitly indicate whether littermates were used or not in the comparison of wt
and ko mice. This is very important because the (gut) microbiota and microbiome composition is
known to affect susceptibility to influenza virus infection.”
Response: As suggested, we have explicitly indicated that wild-type mice used in this study were
not littermates of gene-deficient mice (revised manuscript, page 16, paragraph 3, lines 2-3).
Referee: (Minor remarks) “It is hard to follow the figure numbering in the text. E.g. Fig 3D appears
at the end of the results section. Please use ascending figure numbering in the text.”
Response: We agree with the Referee. We have moved Fig. 3 of the original manuscript to Fig. S4
in supplementary data of the revised manuscript to keep the order of figures.
2nd Editorial Decision
11 January 2016
Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have
now received the enclosed reports from the referees that were asked to re-assess it. As you will see
the reviewers are now globally supportive and I am pleased to inform you that we will be able to
accept your manuscript pending editorial final amendments.
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Please submit your revised manuscript within two weeks.
I look forward to reading a new revised version of your manuscript as soon as possible.
***** Reviewer's comments *****
Referee #1 (Remarks):
The authors have addressed the majority of the prior critiques satisfactorily.
Referee #2 (Comments on Novelty/Model System):
Compared to the original submission, this revised manuscript has improved considerably in
technical quality. More controls are now included, infections were with different influenza A virus
strains and doses and the quality of the micrographs has been improved. The indication that OX40L
is a putative receptor for influenza A viruses is well substantiated.
Referee #2 (Remarks):
The experiments performed for the revision are well performed and strengthen the paper. It is a pity
that immunostaining of the clinical sample could not be improved and had to be withdrawn.
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an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
the exact sample size (n) for each experimental group/condition, given as a number, not a range;
a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
a statement of how many times the experiment shown was independently replicated in the laboratory.
definitions of statistical methods and measures:
 common tests, such as t-­‐test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-­‐Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section;
 are tests one-­‐sided or two-­‐sided?
 are there adjustments for multiple comparisons?
 exact statistical test results, e.g., P values = x but not P values < x;
 definition of ‘center values’ as median or average;
 definition of error bars as s.d. or s.e.m. USEFUL LINKS FOR COMPLETING THIS FORM
http://www.antibodypedia.com
http://1degreebio.org
http://www.equator-­‐network.org/reporting-­‐guidelines/improving-­‐bioscience-­‐research-­‐reporting-­‐the-­‐arrive-­‐guidelines-­‐for-­‐repor
http://grants.nih.gov/grants/olaw/olaw.htm
http://www.mrc.ac.uk/Ourresearch/Ethicsresearchguidance/Useofanimals/index.htm
http://ClinicalTrials.gov
http://www.consort-­‐statement.org
http://www.consort-­‐statement.org/checklists/view/32-­‐consort/66-­‐title
http://www.equator-­‐network.org/reporting-­‐guidelines/reporting-­‐recommendations-­‐for-­‐tumour-­‐marker-­‐prognostic-­‐studies-­‐rem
http://datadryad.org
http://figshare.com
http://www.ncbi.nlm.nih.gov/gap
http://www.ebi.ac.uk/ega
http://biomodels.net/
http://biomodels.net/miriam/
http://jjj.biochem.sun.ac.za
http://oba.od.nih.gov/biosecurity/biosecurity_documents.html
http://www.selectagents.gov/
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
Please ensure that the answers to the following questions are reported in the manuscript itself. We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects. In the pink boxes below, provide the page number(s) of the manuscript draft or figure legend(s) where the information can be located. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable).
B-­‐ Statistics and general methods
Please fill out these boxes  (Do not worry if you cannot see all your text once you press return)
1.a. How was the sample size chosen to ensure adequate power to detect a pre-­‐specified effect size?
Page 22.
1.b. For animal studies, include a statement about sample size estimate even if no statistical methods were used.
Page 22.
2. Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre-­‐established?
NA. Any samples or animals were not excluded from the analysis.
3. Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, please describe. Page 16. For animal studies, include a statement about randomization even if no randomization was used.
Page 16.
4.a. Were any steps taken to minimize the effects of subjective bias during group allocation or/and when Page 21.
assessing results (e.g. blinding of the investigator)? If yes please describe.
4.b. For animal studies, include a statement about blinding even if no blinding was done
Page 17.
5. For every figure, are statistical tests justified as appropriate?
Yes, they are.
Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to Page 22.
assess it.
Is there an estimate of variation within each group of data?
Page 22.
Is the variance similar between the groups that are being statistically compared?
Page 22.
C-­‐ Reagents
6. To show that antibodies were profiled for use in the system under study (assay and species), provide a Page 18.
citation, catalog number and/or clone number, supplementary information or reference to an antibody validation profile. e.g., Antibodypedia (see link list at top right), 1DegreeBio (see link list at top right).
7. Identify the source of cell lines and report if they were recently authenticated (e.g., by STR profiling) and Page 18.
tested for mycoplasma contamination.
* for all hyperlinks, please see the table at the top right of the document
D-­‐ Animal Models
8. Report species, strain, gender, age of animals and genetic modification status where applicable. Please Page 16.
detail housing and husbandry conditions and the source of animals.
9. For experiments involving live vertebrates, include a statement of compliance with ethical regulations and identify the committee(s) approving the experiments.
Page 17.
10. We recommend consulting the ARRIVE guidelines (see link list at top right) (PLoS Biol. 8(6), e1000412, We have confirmed the compliance.
2010) to ensure that other relevant aspects of animal studies are adequately reported. See author guidelines, under ‘Reporting Guidelines’. See also: NIH (see link list at top right) and MRC (see link list at top right) recommendations. Please confirm compliance.
E-­‐ Human Subjects
11. Identify the committee(s) approving the study protocol.
NA
12. Include a statement confirming that informed consent was obtained from all subjects and that the NA
experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.
13. For publication of patient photos, include a statement confirming that consent to publish was obtained.
NA
14. Report any restrictions on the availability (and/or on the use) of human data or samples.
NA
15. Report the clinical trial registration number (at ClinicalTrials.gov or equivalent), where applicable.
NA
16. For phase II and III randomized controlled trials, please refer to the CONSORT flow diagram (see link list NA
at top right) and submit the CONSORT checklist (see link list at top right) with your submission. See author guidelines, under ‘Reporting Guidelines’. Please confirm you have submitted this list.
17. For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines NA
(see link list at top right). See author guidelines, under ‘Reporting Guidelines’. Please confirm you have followed these guidelines.
F-­‐ Data Accessibility
18. Provide accession codes for deposited data. See author guidelines, under ‘Data Deposition’.
NA
Data deposition in a public repository is mandatory for:
a. Protein, DNA and RNA sequences
b. Macromolecular structures
c. Crystallographic data for small molecules
d. Functional genomics data e. Proteomics and molecular interactions
19. Deposition is strongly recommended for any datasets that are central and integral to the study; please NA
consider the journal’s data policy. If no structured public repository exists for a given data type, we encourage the provision of datasets in the manuscript as a Supplementary Document (see author guidelines under ‘Expanded View’ or in unstructured repositories such as Dryad (see link list at top right) or Figshare (see link list at top right).
20. Access to human clinical and genomic datasets should be provided with as few restrictions as possible NA
while respecting ethical obligations to the patients and relevant medical and legal issues. If practically possible and compatible with the individual consent agreement used in the study, such data should be deposited in one of the major public access-­‐controlled repositories such as dbGAP (see link list at top right) or EGA (see link list at top right).
21. As far as possible, primary and referenced data should be formally cited in a Data Availability section. NA
Please state whether you have included this section.
Examples:
Primary Data
Wetmore KM, Deutschbauer AM, Price MN, Arkin AP (2012). Comparison of gene expression and mutant fitness in Shewanella oneidensis MR-­‐1. Gene Expression Omnibus GSE39462
Referenced Data
Huang J, Brown AF, Lei M (2012). Crystal structure of the TRBD domain of TERT and the CR4/5 of TR. Protein Data Bank 4O26
AP-­‐MS analysis of human histone deacetylase interactions in CEM-­‐T cells (2013). PRIDE PXD000208
22. Computational models that are central and integral to a study should be shared without restrictions NA
and provided in a machine-­‐readable form. The relevant accession numbers or links should be provided. When possible, standardized format (SBML, CellML) should be used instead of scripts (e.g. MATLAB). Authors are strongly encouraged to follow the MIRIAM guidelines (see link list at top right) and deposit their model in a public database such as Biomodels (see link list at top right) or JWS Online (see link list at top right). If computer source code is provided with the paper, it should be deposited in a public repository or included in supplementary information.
G-­‐ Dual use research of concern
23. Could your study fall under dual use research restrictions? Please check biosecurity documents (see NA
link list at top right) and list of select agents and toxins (APHIS/CDC) (see link list at top right). According to our biosecurity guidelines, provide a statement only if it could.