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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): expertise in imaging metabolism 

This paper describes a comprehensive and sophisticated study which shows that the low pH in 
lymph nodes (LN), while not inhibiting T cell activation does inhibit the release of some cytokines, 
protecting the LN from premature release of these cytokines. The low pH is shown to be mediated 
by glycolytic lactic acid production by the T cells, which is feedback inhibited by lactate and H+ 
ions in the extracellular space. The mechanism by which inhibition of glycolytic flux inhibited 
cytokine release was not determined, although some potential mechanisms were ruled out. The is 
a publishable story. I have only one, relatively minor, criticism. 

The authors argue that the failure of LPS treatment to alter LN pH is consistent with a robust pH 
stat mechanism, whereby an enlargement of the LN does not disrupt this pH stat. However, no 
evidence is provided that the LNs are enlarged. Also, presumably, LPS would lead to T cell 
activation (and activation of other immune cells in the LN?), increasing glycolytic flux and lowering 
pH, offsetting any increase in LN volume? In summary, I did not find this compelling in vivo 
evidence for their pH stat mechanism. Similarly, they argue that the failure of their bicarbonate 
infusion experiment to raise LN pH was also evidence for the pH stat mechanism in vivo. To 
provide conclusive proof for this they would need to determine the effect of bicarbonate on 
glycolytic flux in the lymph node, according to their mechanism bicarbonate should stimulate 
glycolytic flux. I accept that these are difficult experiments to do (maybe micro dialysis in animals 
administered with isotope-labelled glucose?) but without them one can propose alternative 
explanations for their results without invoking the pH stat mechanism, for example that insufficient 
bicarbonate gets to the LN and therefore cannot elevate pHe. What happens to pHe in the LN of 
nude mice, which have no T cells and reduced acidity, following bicarbonate administration? This 
would be a simpler experiment to do than trying to measure glycolytic flux in the LN 

Reviewer #2 (Remarks to the Author):expertise in T cell signalling and metabolism 

The authors described an acidic niche within the immune system that regulates T cell activation. 
While the model is interesting, the current data presented are not sufficient to support the models, 
and more substantial results are required. 
1. The authors proposed a key role of T cells in affecting the acidic environment in the lymph node. 
Although they used athymic mice to evaluate the involvement of T cells, this system is not 
conclusive as it can be influenced by long-term effects of T cell deficiency. The authors should 
apply acute depletion of T cells to test the direct role of T cells. Also, they need to examine organs 
other than the lymph node to ascertain the specificity of the observed phenomena. 
2. The authors proposed the importance of lactate produced by activated T cells in affecting the 
acidic niche, but the data are entirely based on in vitro observations. It is important to note that 
antigen-specific T cells represent a very small subpopulation of T cells, and it is unclear if lactate 
produced by such antigen-specific T cells in an immune response in vivo is sufficient to alter the 
pH of the lymph node. The authors need to rigorously examine the role of activated T cells in vivo. 
3. The authors proposed that the effect of dampened T cell activation from the acidic niche is to 
prevent tissue damage of the host organ, but there is no data presented. These results are 
essential to establish the physiological relevance of the model. 

Reviewer #3 (Remarks to the Author):expertise in lymphatic system and mathematical modelling 

Regulation of immunity is still poorly understood, and its importance has been underscored by 
recent advances in tumor immunotherapies. The authors present a study analyzing lymph node pH 
and its effect on T cell biology and metabolism. They conclude that T cells create and maintain an 
acidic niche in the lymph node that is self-regulating and which suppresses effector cell function. 

The presented analyses of T cell glycolosis, intracellular and extracellular pH, and cytokine 
production –both in vitro and in vivo – are thorough and convincing. It is clear that pH can affect 
the phenotype of these cells. However, the central thesis of the work – that effector cell function is 
limited in lymph nodes, but enabled upon exodus – is not consistent with known regulation of 
immunity, and conflicts with other data. Most importantly, the authors seem to confound effector 
cell activation and proliferation (which should happen in the lymph node) with effector cell 
cytotoxic activity (which should happen in the target tissue). This is a fundamental flaw in 
rationale that draws into question the relevance of the work. 

Specific comments: 

1. Many tissues have baseline acidic pH, and wound beds are often acidic. Evolutionarily, it would 
be disadvantageous to disable the adaptive immune response in any acidic environment. There is 
actually evidence that acidic wounds heal faster. Can the authors reconcile this with their findings? 

2. 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. 

3. Similarly, the authors suggest that low pH in the node promotes differentiation to a memory 
phenotype in T cells. However, memory T cells are thought to form in the target tissue at the end 
of the immune response. These cells should not be traveling back to the lymph node before 
differentiating into memory cells. 

The most important aspect of effector cell function is not analyzed here. While cytokine production 
is important for effector cell activation and proliferation, cell killing determines the efficacy of the 
immune response. It would make sense that cytotoxic functions are suppressed in the nodes, but 
this is not discussed or analyzed. 

4. The authors write “T-cells activated by antigen-presenting cells (e.g. DCs) produce an acidic 
environment....” This should be shown in vivo, in addition to the in vitro experiments. It is not 
clear that the lymph nodes in Figure 1 were involved in antigen -induced activation. If not, then 
the results show that even quiescent nodes are acidic. 

5. The authors write that “once outside the acidic LN, effector functions of egressing T-cell become 
rapidly uninhibited.” Do the authors think this happens in the blood stream? Or during transit 
through the lung? Or only at the infection site? It seems like we would need other control 
mechanisms to make sure it happens only in the target tissue. 

6. The success of anti-PD1 and anti_PDL1 immunotherapies in cancer shows that it is possible to 
activate effector cell function in tumors, presumably even in the acidic tumor environment. Can 
the authors reconcile this discrepancy? 

Minor comments: 

7. The author should provide more information about the mechanism of how pHLIP responds to pH. 

8. Why was the pimonidazole injected IP rather than into the footpad or i.v.?