January 8, 2014
Dr. Sven Poppert, Associate Editor
Ms. Philippa Harris, Executive Editor
BMC Infectious Diseases
BioMed Central
236 Gray's Inn Road
London WC1X 8HB, United Kingdom
Dear Dr. Poppert,
This letter concerns our revised submission of MS: 1540810597208125, which
previously was entitled, “B cells are competent antigen presenting cells in Leishmania
(Viannia) infection”. We have responded to the reviewers’ comments (specific
responses below). As indicated in your letter, this required time and consequently, this
revision is being submitted after the normal time period, as a new submission, now
entitled, “CD4 T cell activation by B cells in human Leishmania (Viannia) infection” as a
consequence of these revisions.
Specific Responses:
Reviewer: Simona Stager
-“Upregulation of CD69 and CD25 can also occur following exposure to
inflammatory cytokines and does not necessary mean that there is TCR engagement
and antigen recognition. In order to rule out bystander activation, the authors should
perhaps incubate T cells from CL patients with supernatants from B cells (CL
patients).
This is a pertinent observation and we appreciate that this issue has been raised.
In response, we have included additional results that provide a description of the
cytokine profile of supernatants from cultures of purified patient B cells incubated with
pLAg. No significant secretion of 13 cytokines (IFN-, IL-10, IL-4, IL-9, IL-13, IL-6, IL12p70, IL-1β, IL-5, IL-2, IL-17A, IL-22, TNF-) was observed, suggesting that
inflammatory cytokines produced by B cells do not account for CD4 T cell activation
that is observed under these experimental conditions. These data have been added to
the paper and are presented in Figure 6 and summarized briefly (page 13) in the Results
section. Therefore, we consider it is likely that antigen presentation is the main
mechanism of CD4 T cell activation. However, we recognize that direct observation of
cognate B cell-CD4 T cell interactions has not been made. Consequently, the text has
been thoroughly revised to reflect this point. Based on the evidence presented and the
extensive literature establishing B cell antigen presentation as an important mechanism
for T cell activation in several contexts, it is reasonable to state the likelihood of this
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mechanism in our model. Therefore, we have re-written every section and modified the
title to reflect that antigen presentation by B cells in our model is likely, though not
explicitly proven.
We believe that the additional data showing that the supernatants from the pLAg
stimulated B cells do not produce cytokines that might act upon T cells address the
concern raised by the reviewer.
-“Fig.2: representative histograms for the healthy subjects should also be shown.”
We have added representative histograms for the healthy subjects in this figure
(Fig. 2A).
-“What happens to B cells from HS if they are stimulated with pLAg? Do they also
upregulate CD86?”
B cells from healthy subjects do not upregulate any of the activation markers
tested after incubation with pLAg. This information was presented in figure 2B, as
pLAg stimulated B cells had identical levels of expression of activation markers as
controls (non-pLAg-stimulated B cells); this result was previously presented as 100% of
control. . We agree that more clarity was needed on this point and thus have modified
the figure to line graphs that show the lack of change in each of the healthy subjects
(Fig. 2B).
-“Fig.3: without the comparison between the Ag-presenting capacities of B cells and
DCs it is impossible to understand the role of B cells in Ag-presentation. And the
results presented in Table 1 do not allow drawing conclusions on this issue.”
Dendritic cells are potent antigen presenting cells (APCs) that reside in sites of
antigen entry and thus are the main initiators of adaptive immune responses. After
initial activation and expansion of antigen-specific immune cells, B cells have been
shown to be highly efficient APCs and cytokine-secreting cells that can contribute to the
maintenance and shaping of these responses (references 31-37). Our study compares the
efficiency of purified B cells vs. all cells present in peripheral blood mononuclear cells,
which includes DCs, to activate CD4 T cells. We believe this is a valid experimental
design and comparison for human patient studies to establish B cell capacity for this
function. Demonstrating this in humans naturally infected with the parasite is
important to validate this function as a target for immunomodulation and to generate
hypotheses exploring their role in the pathogenesis of reactivation or mucosal
dissemination. We firmly believe the implications of activation of CD4 T cells by B cells
are independent of the capacity that purified DCs would have as APCs in our model.
The issues with Table 1 are addressed below (Page 4 of Response to Reviewers).
-“pLAg should be checked for LPS contaminants”
We believe LPS contamination is not a concern for these studies for two reasons:
1. PBMCs from healthy subjects stimulated with pLAg did not show any CD4 T cell
or B cell activation in relation to controls (Figures 1 and 2).
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2. TLR4 is not expressed by human B cells or CD4 T cells, so co-cultures of these
purified populations do not respond to LPS. Any expression of TLR4 in these cell
types induced by inflammatory signals would result in significant upregulation
of activation markers and/or cytokine secretion in purified T cells or B cells
incubated alone with pLAg if LPS-initiated signals would be present. This was
not the case as shown in figures 4, 5 and 6.
-“what is the difference in terms of phenotype between PBMC B cells from HS and
CL patients?”
There were no differences in the surface molecules expressed by B cells in control
cultures from healthy subjects and CL patients evaluated in this study, namely CD20,
HLA-DR, CD80 and CD86. Representative plots to show this are now included in
Figure 2A.
-“Fig.3: the results for HS B cells should also be shown. Can HS B cells also activate T
cells in the presence of pLAg?” (Note: This is now Figure 4)
Cells from healthy subjects did not show any measurable upregulation of
activation markers in either CD4 T cells or B cells in PBMC cultures. This is in line with
previous observations we have made in these particular populations, where we have
found cells from healthy donors do not proliferate, produce IFN-, TNF- or IL-13, or
upregulate the transcription factors T-bet or GATA-3 in response to stimulation with
live or killed L. panamensis (references 4 and 40 from the paper and Diaz, et al., J Inf Dis
2010, 202:406-415). It is very likely that the absence of clonal expansion of Leishmaniaspecific lymphocytes in non-sensitized subjects is the reason for this lack of response.
The fact that healthy subjects failed to respond to pLAg was previously indicated on
page 10 of the text.
-“Fig.3 (Current Fig. 4): CD69 expression did not increase in most of the samples.”
CD69 expression increased in all of the cultures when comparing B cells, CD4 T
cells plus pLAg to the controls. The increase in some of the patients was of low
magnitude and thus it was hard to observe because of the scale used in the figure. This
scale was used because some of the patients had much larger increases. Such a wide
range of variation (107.3 to 957.6% of control) is not unusual in human studies. We have
modified the scale used in the figure (now Fig. 4C) to make the increase more evident.
-“Moreover, CD69 is upregulated on T cells in the presence of pLAg only. Why?”
CD69 is upregulated on T cells in the presence of pLAg in 7 out of 10 patients. Six
of these patients have a low increase (from 2 to 29%) and one has an 84% increase. The
paired analysis for the whole group did not detect a statistically significant difference
between T cells exposed to pLAg and those not exposed to antigen (p=0.08). We believe
the CD69 upregulation observed in T cells of these patients is due to a TCR independent
mechanism, but considering the lack of statistical significance of the whole group, we
thought this did not warrant a discussion. This upregulation of CD69 is now clearly
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stated in the Results section (page 12). It is important to observe that all patients in
whom CD69 expression was induced by pLAg in CD4 T cells alone demonstrated
further and significant upregulation of this expression in the presence of B cells.
- Representative plots for the CD69 and CD25 staining should be shown.”
Representative plots for CD25 and CD69 expression have been added (Fig. 4A).
-“Table 1: this experiment is quite confusing. Lots of cells in the PBMC can produce
many cytokines during 5 day incubation. It would have been better to measure
cytokine production in B cell/CD4 cultures from HS and CL patients with or without
pLAg.”
We thank the reviewer for pointing out the lack of clarity in this section. Our goal
was to establish that the cytokine profile in both types of cultures (PBMCs and purified
B cells+ CD4 T cells) were similar. We agree with the fact that the display of cytokine
data in Table 1 was confusing, as was the corresponding section in the text. We have
eliminated Table 1 and reorganized the paper to include cytokine data for each culture
type separately and with line figures for all 13 cytokines evaluated (Figure 3 for PBMC
cultures and Figure 6 for CD4 T cell/B cell cultures).
Our previous studies in patients and residents from this region have shown that
no significant cytokine secretion is detected in cultures from healthy subjects (references
39-41). Consistent with this, we did not observe any upregulation of activation markers
in CD4 T cells, B cells or DCs from healthy subjects in this study. Therefore, it would be
expected that there would be little/no cytokine secretion by purified B cells and CD4 T
cells from healthy subjects stimulated with pLAg. Indeed this is what occurs. We have
now included cytokine data from control cultures in Figure 6 (CD4 T cells alone, CD4 T
cells + pLAg, B cells alone and B cells + pLAg) to further clarify this this point.
-“Fig.5: representative plots for the staining should be shown;”
We now include the representative FACS plots (Fig. 7A) showing that L.
panamensis antigen upregulates costimulatory and MHC molecules and enhances BCRmediated endocytosis (Fig. 7C) in Ramos cells.
-“p16, second last paragraph: Ramos cells cannot be compared with B cells from CL
patients, which are heterogeneous and are derived from an inflammatory
environment. It is impossible to draw conclusions about the role of the BCR based on
the experiment shown in Fig. 5A”
We concur that this conclusion was inadequate. Consequently, we have removed
these comments and rewritten our conclusion for this experiment, which now indicates
that L. panamensis can induce upregulation of activation markers without BCR
engagement (page 15).
-“IL-10 is not a Th2 cytokine as stated on p.4 and p.15”
We have changed this statement. Indeed, we agree that numerous other cells can
produce IL-10.
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-“representative plots for the data represented in Fig.2 should be shown”
We have added the representative FACS plots for the expression of CD86, CD80,
HLA-DR for HS and CLP; these are now shown in Figure 2A.
-“the citation #22 on p.5 is incorrect. This work did not directly demonstrate that B
cell present antigens; the conclusions were a suggestion on very indirect evidence.”
We agree with the reviewer on the lack of direct evidence for antigen
presentation in this paper. The study in reference employs an elegant model with B cell
deficient mice and B cell and antibody transfer to conclusively show that a B cell
function different to from antibody secretion mediates CD4 T cell activation
(proliferation and cytokine production). The authors do not show cognate interactions
between Leishmania specific-B cells and CD4 T cells. Since the paper did not include
direct evidence for antigen presentation we have removed this reference from the
introduction and rewritten the sentence in relation with the other study referenced on L.
amazonensis infection.
Reviewer: Ingeborg Becker
-“This conclusion is overstated, since the authors do not show data to validate this
assumption, including controls that rule out the possibility that cytokines produced
by B cells could be responsible for the activation of T cells. It is therefore necessary,
that in addition to showing up-regulation of co-stimulatory molecules in B cells
incubated with pLAg, the cytokine production of these B cells should be analyzed.”
We accept the reviewers’ comment about overstating the conclusion since we
have not shown cognate B cell-CD4 T cell interactions in our cultures. We have added
the data she requests showing that B cells incubated with pLAg do not secrete any
cytokines, eliminating the possibility that cytokines produced by B cells are responsible
for the activation of CD4 T cells. We believe this additional data allows us to suggest
that antigen presentation is a potential mechanism of CD4 T cell activation.
-“It would also be recommendable to wash antigen stimulated B cells before
incubating them with CD4 T cells.”
We thank the reviewer for this suggestion. If washed cells would not activate
CD4 T cells, this experiment would suggest that CD4 T cells are being activated by
pLAg directly through a TCR-independent mechanism without participation of any
APC. We show that CD4 T cells incubated alone with pLAg do not upregulate
activation markers (Figure 4; Page 11 Results Section); further, we have added data
showing that B cells stimulated with pLAg also do not secrete cytokines (Figure 3;
Please also see response above to Dr. Stager). Furthermore, CD4 T cells from healthy
subjects do not show any activation after incubation with pLAg, also proving that pLAg
does not activate CD4 T cells through TCR independent mechanisms. Therefore, the
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proposed experiment would be redundant in terms of ruling out activation of T cells by
pLAg alone, and would not show occurrence of cognate B cell-CD4 T cell interactions.
-“Furthermore, control experiments that rule out direct B and T cell contact, as can be
achieved by Boyden Chambers, should be included.”
We have addressed this point through the additional experiments added
concerning evaluation of the production of 13 cytokines by B cells stimulated with
pLAg. These data clearly show a lack of secretion of cytokines by B cells after
stimulation with pLAg; therefore cytokine secretion does not appear to be the
mechanism responsible for CD4 T cell activation. We agree that the experiment
proposed with Boyden chambers provides an alternate approach and could further
support this point. However, the lack of cytokine secretion is compelling and the
proposed experiment would not show any cognate B cell-CD4 T cell interactions.
-“In conclusion, the authors must either provide uncontroversial evidence showing
that B cells are competent antigen presenting cells, or adjust the title and all the
sections referring to the B-cell antigen presenting capacity and substitute them with a
statement only describing the up-regulation of co-stimulatory molecules in B cells.”
The title has been modified to “CD4 T cell activation by B cells in human
Leishmania Viannia infection” and the text has been revised to indicate activation of CD4
T cells by B cells rather than antigen presentation. However, we believe that after
addition of the cytokine data it is reasonable for us to suggest that antigen presentation
has a role in CD4 T cell activation based on the following: 1) we show that L. panamensis
antigens induce CD4 T cell activation only in individuals having Leishmania infection, a
result reflecting the occurrence of an adaptive immune response to Leishmania antigens
and implicating specific CD4 T cell clones expanded in vivo and therefore TCRmediated activation; 2) the vast literature showing the competence of B cells as APCs; 3)
the fact that B cells are the only APCs present in the B cell/CD4 T cell co-cultures; and
4) the lack of cytokine production by B cells when incubated with pLAg that eliminates
the possibility of bystander activation.
Despite this evidence, to provide an uncontroversial demonstration of antigen
presentation we would have to show cognate B cell-CD4 T cell interactions directly.
This would require us to identify and isolate Leishmania-specific B cells and CD4 T cells
from the patients to establish cell lines. This technology is not within our current
capacity and is beyond the scope of this study. Therefore, as the reviewer suggests, the
paper has been re-written with this limitation clearly indicated and modified the title to
reflect that antigen presentation by B cells in our model is likely, but not a proven fact.
-“Reference 30 must be revised, since it does not describe how pLAg was prepared
(the manuscript section on Cell Culture states that reference 30 discloses how pLAg
was produced, yet this is not evidenced).”
We thank the reviewer for pointing out this inconsistency. We have replaced the
reference with a brief description of the preparation of pLAg (Page 8, Methods section;
Cell Culture).
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Overall, we believe that our revised manuscript entitled, “CD4 T cell activation
by B cells in human Leishmania (Viannia) infection” as a consequence of these revisions,
is now considerably improved. Our findings clearly demonstrate the importance of B
cells in regulating the immune response during L. (Viannia) infection, indicating that
these cells provide a target for immunotherapeutic approaches for treatment.
Sincerely,
Diane McMahon-Pratt
Daniel Rodriguez-Pinto
Nancy Saravia
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