Review for 10.1186/1471-230X-13-37 (2013)

Reviewer's report
Title:Attenuated antigen-specific T cell responses in cirrhosis are accompanied
by elevated serum interleukin-10 levels and down-regulation of HLA-DR on
monocytes
Version: 2Date:27 November 2012
Reviewer: Pilar Garcia-Peñarrubia
Reviewer's report:
The manuscript by Jack Peter and colleagues entitled: “Attenuated
antigen-specific responses in cirrhosis are accompanied by elevated serum
interleukin-10 levels and down-regulation of HLA-DR on monocytes” aims to
evaluate the cellular immune status in cirrhotic patients related with the presence
or absence of inflammatory processes. To accomplish this goal they determined
antigen-specific T cell response towards EBV derived peptides EBNA1 and
BZLF1 from two groups of cirrhotic patients (without inflammation vs
inflammation, according to CRP lower or higher than 10 mg/ml, respectively). As
control group they used peripheral blood leukocytes from healthy donors.
Although the topic is potentially interesting, the investigation suffers from a basic
flaw in a coherent analysis and presentation of the results according to the
proposed aims. The manuscript is flawed for the following reasons:
Mayor compulsory revision points:
1) After stimulation with the peptide pools from the viral antigens EBNA1 and
BZLF1, the number of responding T cells and the amount of TNF-# or IFN-#
production did not differ between the three pre-defined groups (Fig 4A, although
in the corresponding results section is referred to as Fig.3A (page 9 in the
manuscript)). Furthermore, differences in the fraction of patients with a
null-response towards the EBNA1 and BZLF1 peptides were also non significant
(9 patients (41%) with inflammation compared to 3 (23%) without inflammation,
and 7 patients (32%) with inflammation compared to 3 (23%) without
inflammation, respectively).
2) Nevertheless, they found significant higher serum IL-10 levels in cirrhotic
patients null-responders to EBV peptides. Related to this, they stated that
“Cirrhotic null-responders to EBNA1 or BZLF1 had higher IL-10 levels than
cirrhotic patients with at least one positive response (p=0.048 and p=0.008,
respectively) (Figure 4C)”. However, this figure shows that the production of
IL-10 is significantly higher only between boxes 1 and 3. It is unclear what
differences are being compared, and how many patients are included in every
group. Additionally, the authors did not mention whether the cirrhotic patients
included in the IL-10 null-responding analysis are cirrhotic in general, or they are
separated by inflammation or non-inflammation status. If the analysis were
performed on all the non-responding cirrhotic patients, the rest of the manuscript
about the analysis of CRP, LPB, DR and CD25 expression etc, is devoid of
interest, because these observations have been described in the cirrhosis
literature.
3) Related to SEB stimulation, the authors refer that “the fraction of
IFN-#-producing or TNF-#-producing CD4+ and CD8+ cells differed significantly
between the three groups and was lower in cirrhotic patients with inflammation
than in cirrhotic patients without inflammation”. However, these data are not
displayed as a figure and the described percentages did not even show the SD
or SEM. Furthermore, SEB is not antigen-specific, since it binds to both MHC-II
molecules and TCR from all the CD4+ T cells expressing determined V# regions.
Thus, it is a polyclonal non-specific T cell stimulation, which is not the objective of
this work. Thus, it could be rather shown as a positive control of T cell response.
Additionally, a negative control of the antigen-specific T cell response should be
shown, i.e., using EBV peptides in the absence of anti-CD28 and anti-CD49d.
4) They stated that “LBP and IL-10 levels differed significantly between the three
groups and were highest in patients with cirrhosis and inflammation (Figure 1a
and 1b)”. In this context, P-values obtained from the Kruskal-Wallis test indicate
inequality mainly because of the small response of the healthy control group.
However, when using appropriated post-hoc Dunn’s, the asterisks show that
differences in LBP and IL-10 levels are only significant when compared to the
healthy controls, but they are not significant between the two established cirrhotic
groups. The same problem can be observed in figures 2A, 2B, 3A and 3B, and
with the P values shown in Table 1.
5) Despite searching for immune T cell specific differences between groups of
cirrhotic patients according to the presence or absence of inflammation, the
analysis of cell populations was not elaborate. For example, information about
the activation status other than CD25 or chemokine receptor status of the cells
could have been provided. Other cell types, such as Treg populations could have
also been provided.
Minor essential revisions:
1) CRP levels in the control group are missing as it is the method used to
determine CRP levels.
2) In page 5 authors say that “Lymphocyte and monocyte populations were
identified by the use of forward and right angle light scatter and by CD14, CD4 or
CD8, respectively”.
CD4 or CD8 must be named before CD14 according to the term: respectively.
3) In ref. 28, EBV peptides were used at doses of 3,5 and 1 micrograms/ml, while
in this work they used 0,6 ngrams/ml. This fact could explain the poor results
obtained, especially in the absence of negative controls.
3) Reference 27 is incomplete and the year is 2012 instead of 2011. There is
also another important contribution on the CRP subject in cirrhosis that has not
been cited here (Cervoni et al., 2012 J Hepatol. 56:1299-2304).
5) Use of grammar should be checked.
Level of interest: An article of insufficient interest to warrant publication in a
scientific/medical journal
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.