Reviewer's report
Title:Longitudinal fluctuations in PD1 and PD-L1 expression in association with
changes in anti-viral immune response in chronic hepatitis B
Version: 1Date:9 April 2012
Reviewer: Christoph Neumann-Haefelin
Reviewer's report:
Wenjin and co-workers study PD1 and PD-L1 expression during flares of chronic
HBV infection. They demonstrate that inflammatory flares are associated with a
reduction of viral load and HBsAg levels on the one hand, and strongly increased
HBV-specific CD8+ T cell responses as well as high PD1 and PD-L1 expression
on the other hand. While HBV-specific CD8+ T cell responses as well as PD1
expression decline as the flares resolves, intrahepatic PD-L1 expression remains
relatively high. The authors argue that this inhibitory milieu at the site of infection
may facilitate persistent HBV infection.
Although generally of interest, there are several issues that need attention prior
to publication:
Major compulsory revisions:
1. Quality of the HBV-specific pentamer stainings: HBV-specific CD8+ T cell
responses are usually extremely week in chronic infection, even when tetramers
or pentamers are used for detection. Multimer-positive cells, however, usually
separate as distinct cell populations. This is definitely not the case in Fig. 1. The
FACS blots that are shown produce several concerns: Why are there nearly no
CD8+ cells in some samples? Why do the multimer-positive cells (if any!) not
separate as distinct cell populations? Are the authors sure that the “positive”
fractions are not rather an effect of incorrect compensation (cells with a high
FITC signal shifting towards PE)? Importantly, how was the “background”
defined, which “never exceeded 0.02%”? The authors need to show a)
Background staining without multimer and b) multimer control stainings from
healthy controls.
2. The authors argue that the relatively high level of intrahepatic PD-L1
expression during the inflammatory regression period facilitates persistent HBV
infection. However, PD-L1 expression declines to a third of its maximum (T2 to
T4). How high is the intrahepatic PD-L1 expression in patients without an
inflammatory flare? Are the authors sure that it is indeed increased during the
regression period compared to time points prior to the inflammatory flare? The
lack of these data are a major limitations of the study, and they need to be
experimentally addressed and/ or carefully discussed.
3. The figures need true legend titles, not just “Representative dot plot from 15
independent experiments” etc. By the way, what does “from 15 independent
experiments” mean? I suppose, one out of 15 patients is shown here.
Minor essential revisions:
1. Materials and methods: subjects: “All patients were… with HBV-DNA levels
greater than 10x6 copies/ml”. This is incorrect. According to the table, 5 patients
have an HBV-DNA level below 10x6 copies/ml.
2. Table in material and methody: units for ALT, HBsAg, HBV-DNA are missing
or incomplete. HLA-A2 has been misspelled.
3. Methods and throughout the manuscript: The authors switch from tetramers to
pentamers. What kind of multimers was used? From which supplier?
4. Page 5: “The frequency of PD1 expression on pentamers was significantly
higher than that among total circulating CD8+ T cells (Fig 2a, Fig 6c)”. The
frequency of PD1+ cells in total circulating CD8+ T cells is not shown in the
figures stated here.
5. Legend to Fig. 2b: Antibodies against pentamer were definitely not used in this
assay.
6. Page 11: “In general, circulating and inta-hepatic PD1 and PD-L1 expression
levels were within the normal range before the initiation of inflammation, and
were progressively uprgulated at the onset of liver inflammation (T1)…”
Intra-hepatic PD1 and PD-L1 expression were not analyzed before the initiation
of inflammation and at T1. As discussed above, this is a major limitation of the
study and needs to be clearly stated.
7. Language issues:
- Page 1, abstraction instead of abstract
- Page 1, hepatitis B infection instead of hepatitis B virus infection
- Page 2, summarized in follow table instead of summarized in the following table
- Page 3, anti-CD8-TITC instead of anti-CD8-FITC
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Longitudinal fluctuations in PD1 and PD-L1 expression in association with
changes in anti-viral immune response in chronic hepatitis B
Version: 1Date:9 April 2012
Reviewer: Christoph Neumann-Haefelin
Reviewer's report:
Wenjin and co-workers study PD1 and PD-L1 expression during flares of chronic
HBV infection. They demonstrate that inflammatory flares are associated with a
reduction of viral load and HBsAg levels on the one hand, and strongly increased
HBV-specific CD8+ T cell responses as well as high PD1 and PD-L1 expression
on the other hand. While HBV-specific CD8+ T cell responses as well as PD1
expression decline as the flares resolves, intrahepatic PD-L1 expression remains
relatively high. The authors argue that this inhibitory milieu at the site of infection
may facilitate persistent HBV infection.
Although generally of interest, there are several issues that need attention prior
to publication:
Major compulsory revisions:
1. Quality of the HBV-specific pentamer stainings: HBV-specific CD8+ T cell
responses are usually extremely week in chronic infection, even when tetramers
or pentamers are used for detection. Multimer-positive cells, however, usually
separate as distinct cell populations. This is definitely not the case in Fig. 1. The
FACS blots that are shown produce several concerns: Why are there nearly no
CD8+ cells in some samples? Why do the multimer-positive cells (if any!) not
separate as distinct cell populations? Are the authors sure that the “positive”
fractions are not rather an effect of incorrect compensation (cells with a high
FITC signal shifting towards PE)? Importantly, how was the “background”
defined, which “never exceeded 0.02%”? The authors need to show a)
Background staining without multimer and b) multimer control stainings from
healthy controls.
2. The authors argue that the relatively high level of intrahepatic PD-L1
expression during the inflammatory regression period facilitates persistent HBV
infection. However, PD-L1 expression declines to a third of its maximum (T2 to
T4). How high is the intrahepatic PD-L1 expression in patients without an
inflammatory flare? Are the authors sure that it is indeed increased during the
regression period compared to time points prior to the inflammatory flare? The
lack of these data are a major limitations of the study, and they need to be
experimentally addressed and/ or carefully discussed.
3. The figures need true legend titles, not just “Representative dot plot from 15
independent experiments” etc. By the way, what does “from 15 independent
experiments” mean? I suppose, one out of 15 patients is shown here.
Minor essential revisions:
1. Materials and methods: subjects: “All patients were… with HBV-DNA levels
greater than 10x6 copies/ml”. This is incorrect. According to the table, 5 patients
have an HBV-DNA level below 10x6 copies/ml.
2. Table in material and methody: units for ALT, HBsAg, HBV-DNA are missing
or incomplete. HLA-A2 has been misspelled.
3. Methods and throughout the manuscript: The authors switch from tetramers to
pentamers. What kind of multimers was used? From which supplier?
4. Page 5: “The frequency of PD1 expression on pentamers was significantly
higher than that among total circulating CD8+ T cells (Fig 2a, Fig 6c)”. The
frequency of PD1+ cells in total circulating CD8+ T cells is not shown in the
figures stated here.
5. Legend to Fig. 2b: Antibodies against pentamer were definitely not used in this
assay.
6. Page 11: “In general, circulating and inta-hepatic PD1 and PD-L1 expression
levels were within the normal range before the initiation of inflammation, and
were progressively uprgulated at the onset of liver inflammation (T1)…”
Intra-hepatic PD1 and PD-L1 expression were not analyzed before the initiation
of inflammation and at T1. As discussed above, this is a major limitation of the
study and needs to be clearly stated.
7. Language issues:
- Page 1, abstraction instead of abstract
- Page 1, hepatitis B infection instead of hepatitis B virus infection
- Page 2, summarized in follow table instead of summarized in the following table
- Page 3, anti-CD8-TITC instead of anti-CD8-FITC
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Longitudinal fluctuations in PD1 and PD-L1 expression in association with
changes in anti-viral immune response in chronic hepatitis B
Version: 1Date:9 April 2012
Reviewer: Christoph Neumann-Haefelin
Reviewer's report:
Wenjin and co-workers study PD1 and PD-L1 expression during flares of chronic
HBV infection. They demonstrate that inflammatory flares are associated with a
reduction of viral load and HBsAg levels on the one hand, and strongly increased
HBV-specific CD8+ T cell responses as well as high PD1 and PD-L1 expression
on the other hand. While HBV-specific CD8+ T cell responses as well as PD1
expression decline as the flares resolves, intrahepatic PD-L1 expression remains
relatively high. The authors argue that this inhibitory milieu at the site of infection
may facilitate persistent HBV infection.
Although generally of interest, there are several issues that need attention prior
to publication:
Major compulsory revisions:
1. Quality of the HBV-specific pentamer stainings: HBV-specific CD8+ T cell
responses are usually extremely week in chronic infection, even when tetramers
or pentamers are used for detection. Multimer-positive cells, however, usually
separate as distinct cell populations. This is definitely not the case in Fig. 1. The
FACS blots that are shown produce several concerns: Why are there nearly no
CD8+ cells in some samples? Why do the multimer-positive cells (if any!) not
separate as distinct cell populations? Are the authors sure that the “positive”
fractions are not rather an effect of incorrect compensation (cells with a high
FITC signal shifting towards PE)? Importantly, how was the “background”
defined, which “never exceeded 0.02%”? The authors need to show a)
Background staining without multimer and b) multimer control stainings from
healthy controls.
2. The authors argue that the relatively high level of intrahepatic PD-L1
expression during the inflammatory regression period facilitates persistent HBV
infection. However, PD-L1 expression declines to a third of its maximum (T2 to
T4). How high is the intrahepatic PD-L1 expression in patients without an
inflammatory flare? Are the authors sure that it is indeed increased during the
regression period compared to time points prior to the inflammatory flare? The
lack of these data are a major limitations of the study, and they need to be
experimentally addressed and/ or carefully discussed.
3. The figures need true legend titles, not just “Representative dot plot from 15
independent experiments” etc. By the way, what does “from 15 independent
experiments” mean? I suppose, one out of 15 patients is shown here.
Minor essential revisions:
1. Materials and methods: subjects: “All patients were… with HBV-DNA levels
greater than 10x6 copies/ml”. This is incorrect. According to the table, 5 patients
have an HBV-DNA level below 10x6 copies/ml.
2. Table in material and methody: units for ALT, HBsAg, HBV-DNA are missing
or incomplete. HLA-A2 has been misspelled.
3. Methods and throughout the manuscript: The authors switch from tetramers to
pentamers. What kind of multimers was used? From which supplier?
4. Page 5: “The frequency of PD1 expression on pentamers was significantly
higher than that among total circulating CD8+ T cells (Fig 2a, Fig 6c)”. The
frequency of PD1+ cells in total circulating CD8+ T cells is not shown in the
figures stated here.
5. Legend to Fig. 2b: Antibodies against pentamer were definitely not used in this
assay.
6. Page 11: “In general, circulating and inta-hepatic PD1 and PD-L1 expression
levels were within the normal range before the initiation of inflammation, and
were progressively uprgulated at the onset of liver inflammation (T1)…”
Intra-hepatic PD1 and PD-L1 expression were not analyzed before the initiation
of inflammation and at T1. As discussed above, this is a major limitation of the
study and needs to be clearly stated.
7. Language issues:
- Page 1, abstraction instead of abstract
- Page 1, hepatitis B infection instead of hepatitis B virus infection
- Page 2, summarized in follow table instead of summarized in the following table
- Page 3, anti-CD8-TITC instead of anti-CD8-FITC
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
assistant