The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells

Decision to publish would be subject to satisfactory responses. In particular, it is essential that you address the following with changes to the manuscript:

(i) For proper presentation to a general audience, it is necessary that you present the existing published literature that is directly relevant to your work in the introduction. This enables your results to be understood in the context of that literature. Your discussion may then concentrate on differences/similarities/extensions of that work by your own data and on whether the overarching question of the role of physiological/pathological signalling in directing T-reg activity has or has not been answered by the literature in total.

We have followed this recommendation and provided additional information on the “state of the art” and previous work covering the topic of our work. This is now included in the Introduction section.

(ii) An important part of the novelty is dependent on restriction of recombination using the Foxp3-Cre-YFP line. More data is needed to show that recombination is indeed restricted as proposed, or that additional sites of recombination might contribute to the phenotype if they exist.

This point is well taken. As previously stated in our previous version, expression of the Cre-allele appears as highly restricted to the Foxp3⁺ subset of lymphoid cells. As expected however, we have found and reported in Figure 8-figure supplement 1 that a minor, but identifiable subset of non-lymphoid (CD45-negative) cells (from 1 to 5%, depending on the organ considered) expresses the Cre allele. Despite our efforts, we could not identify the nature of these cells, and their contribution to the observed phenotype remains therefore to be established. We do however believe that (i) the fact that PHD2-KO Tregs display a reduced suppressive capacity when co-transferred with naïve T cells in Rag2-deficient mice (Figure 4) and (ii) the observations performed in heterozygous Foxp3^Cre/+ Egln1^fl/fl (Figure 3) demonstrating that loss of PHD2 expression affects the Treg phenotype in a cell-autonomous, intrinsic fashion, strongly indicate that PHD2-expression is required for adequate Treg phenotype and function irrespectively of the environment in which they differentiate.

(iii) It is necessary to provide more detailed description of the haemorragic abdomen – in particular, whether this might imply a more complex phenotype affecting other components of haematopoiesis.

We have been able to demonstrate that these mice display an increased haematocrit and enhanced vascular permeability that could be a consequence of enhanced Vegfa expression by Tregs. At present, the mechanism leading to these haematological consequences remain to be established. Notably however, an haemorrhagic abdomen was not observed in all mice examined (albeit in a majority of them, approximatively 70%), whereas the phenotypical alterations of Tregs and the enhanced activation of Tconvs were confirmed in all animals displaying PHD2-deficient Tregs, once again suggesting that the effect of PHD2 on Treg biology are not a consequence of altered haematological parameters.

(iv) All reviewers felt more data was required to support the mechanistic hypotheses. However, it was acknowledged that this would likely demand an unrealistic amount of further work. The authors should therefore either provide any further experiment support they can, or moderate the mechanistic claims. In particular, they should address the issue raised by one of the reviewers that changes in splenic architecture could have confounded interpretation of the importance of lymphocyte mislocalization in that organ.

This point is well taken, and we have now deleted the last figure of the manuscript and provided a less speculative conclusion to our study. We pointed to a possible link between the PHD2-HIF2α axis and STAT1 activation as an important factor regulating Treg phenotype and function.

Reviewer #1 (Recommendations for the authors):

Please add more description of the existing literature describing effects of disruption of HIF-signalling on Treg function to the Introduction and identify (and discuss possible explanations for) similarities and differences between these reports and your own data in the Discussion.

We believe we have adequately responded to this request

Please clarify expression of YFP outside the Treg lineage and any changes in Phd2 expression in non-lymphoid tissues (including endothelium, liver and colon). Please comment on the relative changes in red and white pulp of the spleen and provide data to show whether haematocrit has or has not changed in the Phd2-deficient Treg mice.

We have performed the requested experiments but could not achieve a definite conclusion regarding the cause of the haematological abnormalities observed in PHD2^ΔTregs mice.

Please indicate absolute cell counts as well as percentages in the various lymphoid organs.

An example of absolute cells counts is now provided in the Figure 1—figure supplement 4.

It would be nice if critical gating strategies were shown to support the flow cytometry analyses.

An example of the gating strategy used throughout this study is now provided in Figure 1—figure supplement 3

Availability of raw data.

All raw data are now available, see Excel Source data.

Reviewer #2 (Recommendations for the authors):

Despite this not being necessary for the publication of this manuscript, my only suggestion to the authors is to test effect of chemical PHD inhibitor drugs on TREG function and use the specific HIF2 inhibitor PT-2385 to try and abolish any potential side effect. Additionally, the use of PT2385 per se in any of the in vivo assays with the TREG PHD2 KO cells would not only rule out any cellular adaptation mechanism that might occur during TREG development in double or triple KOs and would be relevant for the clinic as this inhibitor is being tested in humans.

We thank the reviewer for this interesting suggestion that we will take into consideration for our future work

Reviewer #3 (Recommendations for the authors):

1. The authors should analyze the early Treg development in the thymus.

We thank you the reviewer for this insightful suggestion. We have now analysed the frequency of thymic Treg precursors (see Figure 2 b-d) and found an increased frequency of immature Tregs displaying the Foxp3^lo CD25^neg phenotype in the thymus of PHD2^ΔTregs mice. Consistent with the expected tissue distribution of the Cre allele, no differences were found in the frequency of the Foxp3^neg CD25^pos subset of precursor Tregs. Overall, this analysis suggests a role for PHD2 in thymic Treg development and a possible “immature-like” state of peripheral Tregs in PHD2^ΔTregs mice.

2. Extrinsic vs. intrinsic effect should be addressed using Foxp3-cre +/- mice, or co-adoptive transfer approaches.

We thank again the reviewer for this suggestion. The results from these experiments are now depicted in Figure 3, and demonstrate that the phenotypic alterations found in PHD2-deficient Tregs appear as “cell autonomous”. Moreover, this experiment also strongly suggest that PHD2-deficient T cells display reduced fitness when compared to their WT counterpart, a finding that we have been able to confirm in adoptive co-transfer experiments (data not shown).

3. The authors should perform more experiments to clarify the mis-localization vs. direct suppression in vivo. The current model does not explain the effector/TH1-like phenotype.

This comment is well taken, and as previously discussed (see remark n°8), we are now convinced that our work does not provide sufficient arguments to sustain the “mis-localisation” hypothesis, partly due to the unresolved question of the profound changes in spleen cell morphology observed in PHD2^ΔTregs mice. We have therefore omitted this set of data from the present version of the manuscript.

4. More mechanistic experiments are required to address the PHD2-HIF2s relation to the Stat1 phosphorylation, and the mis-localization issue.

We fully agree with this final remark, but the amount of work that would be required to experimentally connect HIF2α to STAT1 activation is probably beyond reach within the time allowed for this review. In particular it appears, from the literature, that STAT1 regulation is a complex phenomenon involving a set of post-translational modifications (including methylation and palmitoylation) that could represent targets of HIF2α-mediated regulation.