Peer review of: T-cells produce acidic niches in lymph nodes to suppress their own effector functions (2020)



 

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

The authors have answered my criticisms and I have no further comments. 

Kevin Brindle 

Reviewer #2 (Remarks to the Author): 

In the revised manuscript, the authors have address part of my previous concerns, although not 
completely. In particular, for the in vivo relevance of T cell activation in affecting the lymph node 
pH, there are still no data supporting the conclusion (my original point #2). The authors should 
use antigen-specific T cells for adoptive transfer experiment, and examine in vivo pH with or 
without antigen stimulation. 
Part of my original question #1 asked for the results from organs other lymph nodes, but this 
question was left unanswered. 
For my original question #3, I understand the difficulty in making a definitive conclusion, but the 
authors should tune down their conclusions and impacts accordingly, to avoid over-interpretation 
of their results and conclusions. 

Reviewer #3 (Remarks to the Author): 

The authors have adequately addressed many of the issues raised in the original review. However, 
there are still a few remaining points that require attention: 

Original comment: The authors show that acidity decreases T cell proliferation and cytokine 
production. But these are the main activities that are supposed to happen in the lymph node. 
Antigen presentation leads to activation and effector cell proliferation locally, before the cells leave 
the node. There are also critical paracrine effects of the cytokines that would be prevented by the 
authors’ proposed mechanism. For example, helper T cells activated in the node produce cytokines 
that activate CD8 T cells and B cells in the same node. In this way, the lymph node is a factory of 
effector cells and antibodies that, if inhibited by acidic pH, would be expected to thwart systemic 
immunity. These issues are not discussed in the manuscript. 

Author response: Our data are not suggesting that acidity decreases T cell proliferation. Indeed, 
Fig S11D showed that proliferation of activated CD4+ and CD8+ T cells was only modestly affected 
by acidity. To investigate this with more sensitive tools, we performed [3H]thymidine incorporation 
assays of naïve (CM), and peptide- or CD3-activated pMel, OT-I and OT-II cells. As shown in the 
figure below (provided for review), while there was more incorporation at pH 7.4, it was not 
substantial, consistent with the flow data of S11D. 

Follow-up comment: 
It is not clear what the authors mean by “modestly” or “substantial”, but there appear to be 
statistically significance differences, and the authors state on lines 354-55: “In addition to its 
inhibitory effect on effector functions, acidosis also decreased the proliferation rate of T-cells.” 

Regarding the other effects of cytokines in the lymph node, the authors did not address this issue. 
I understand that regulation and function of the cytokine milieu in the lymph node is poorly 
understood and difficult to fully dissect in this study. However, the current text gives the 
impression that everything is controlled by pH, and no other mechanisms are needed. This could 
mislead readers. There must be other feedback systems preventing the T cells from mounting an 

immune response within the lymph node. An obvious mechanism is the lack of appropriate target 
cells to kill there (i.e., when there is no intra-node infection). 

Along these lines, the authors should discuss why, in the context of their theory, anti-inflammatory 
cytokines such as IL-4, IL-10 and IL-13 are also down-regulated at low pH (Figure S11B). 

Additional comment: The authors should clarify which T cell “effector functions” they are 
addressing in the study. They are not presenting data on cytotoxic function or degranulation, but 
only production of cytokines (primarily IFgamma). The uninitiated reader might infer that low pH is 
preventing the T cells from actively killing LN bystander cells; but this is not really a danger, as 
there are other control mechanisms in place. The data show that pH is low in the LNs, and that T 
cells produce different cytokine levels at low pH. The authors should focus on this, and the 
potential ramifications of increased IFgamma in the node. Other speculation and language related 
to subsequent T cell effector function should be de-emphasized or removed.