DJ‐1 depletion prevents immunoaging in T‐cell compartments

Abstract Decline in immune function during aging increases susceptibility to different aging‐related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T‐cell subpopulations, still remain largely elusive. Here, we show that loss of DJ‐1 encoded by PARK7/DJ‐1, causing early‐onset familial Parkinson’s disease (PD), unexpectedly diminished signs of immunoaging in T‐cell compartments of both human and mice. Compared with two gender‐matched unaffected siblings of similar ages, the index PD patient with DJ‐1 deficiency showed a decline in many critical immunoaging features, including almost doubled non‐senescent T cells. The observation was further consolidated by the results in 45‐week‐old DJ‐1 knockout mice. Our data demonstrated that DJ‐1 regulates several immunoaging features via hematopoietic‐intrinsic and naïve‐CD8‐intrinsic mechanisms. Mechanistically, DJ‐1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T‐cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ‐1 in regulating immunoaging, discovering a potent target to interfere with immunoaging‐ and aging‐associated diseases.


28th Jun 2021 1st Editorial Decision
Dear Dr. Hefeng, Thank you for the submission of your research manuscript to our journal, which was now seen by three referees, whose reports are copied below.
Referees find the proposed role of DJ-1 in immunoaging in principle interesting. However, they also raise significant concerns that need to be addressed for publication here. In particular, -Functional assays testing the effects of DJ-1 on CD8+ T cells are required to substantiate the claims on reduced immunosenescence (referee #1, major point 2; referee #2, second paragraph).
-Immunometabolic status of T cell populations needs to be better characterized (referee #1, major point 3).
-Immunophenotypic characterization of patients' CD8+ T cells should be presented in a complete form (referee #1, major point 1).
-The effect of DJ-1 depletion on TVM cell population needs to be investigated (referee #2, paragraph 3).
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I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if you have questions or comments regarding the revision. The study by Zeng et al describes as possible role of DJ-1 in regulating the aging of the immune system, by claiming that DJ-1 depletion slows down the aging of T cells compartment of adaptive immunity. The manuscript is interesting and does suggest a possible new role of DJ-1, but in my opinion, in the actual form it fails to prove a functional role of DJ-1 in immunoaging, as it does not provide any real insight on the mechanisms that link DJ-1 KO to slow down immunosenescence. In some cases, the rationale of the proposed experiments is not completely clear Major points.
Thera are four major flaws in this manuscript: -I could not find the "classic" immunophenotypic characterization of patients' CD8+ T cells, i.e. Tn/tcm/Tem/Temra, while it is shown for CD4+ T cells in supplementary data. The authors should show this figure. Is there any difference in the proportion of T cell subsets between the proband, the sibling and age and sex-matched controls?
-The observations on CD8+ T cells are phenotypic but do not demonstrate an actual reduced senescence of T cells. One cannot exclude that these cells are phenotypically "younger" but functionally old. Thus, a functional test is needed on patients' T cells to prove that they are actually less senescent, for example by stimulating T cells in vitro with aCD3/aCD28 followed by division rate quantification via CFSE labeling and analysis of T cell division in T cells subsets.
-The experiments on mitochondria are merely descriptive but do not help understand the role of the organelle on the delayed immunoaging. On the basis of the studies by Pierce et al, which provided evidence of the crucial role of metabolic shift and mitochondrial reorganization in the switttch from Tn to Tm cells, I think it could be much more informative an immunometabolic evaluation of T cell subsets (OCR, ECAR) in patients, as well in mouse models. I suggest performing this experiment before and after T cell stimulation, to clarify if there is an impairment in T cell activation that can explain the lower levels of cells with exhausted or senescent phenotype.
-I do not understand the rationale below the choice of performing BM transplantation. As well established in the past, one of the most important causes of T cell compartment senescence is thymus involution, whose progressive shrinking does not allow proper development of new naïve T cells. Have the authors monitor parameters related to thymic output, such as CD31 expression or sjTREC+ T cells in aged mice? Without this element, it is hard to understand if the effect of DJ-1 is a general effect on the immune system or a specific effect on T cells.
Minor points -I strongly suggest not to overlap the use of exhausted and senescent throughout the manuscript. Although overlapping, the two concepts are clearly different, as well described by Xu and Larbi (2017), and sliding from one to the other does not help the comprehension of the message.
-Since I do not see the presence of CD3 Ab in the list of Abs used for analysing human T cells, I think it is necessary to show the gating strategy for the immunophenotype. -There are some typos in the text. Please be consistent in how molecules are named in the text (for example IFN-g, not IFNg -I understand that we are in COVID-19 pandemic era, but I think it is superfluous to cite COVID-19 in a paper that has nothing to do with it.
Referee #2: The study begins with an index case containing a homozygous DJ-1 mutation that has driven early onset Parkinson's disease. Previous work had shown that DJ-1 mutation resulted in diminished Treg cells in aged but not young mice. Further analysis of an aged phenotype in this individual revealed a reduced frequency of senescent CD4 and CD8 T cells, reduced expression of markers associated with senescence, differentiation or exhaustion, and reduced TCR narrowing, compared to two unaffected siblings. To further probe the DJ-1 deficiency, global DJ-1 KO mice were used. Similar to the human phenotype, DJ-1 KO mice show a retention of Tn cells and a reduction of Tem cells with age and a general reduction in markers and characteristics associated with aging, i.e. markers of exhaustion and senescence, and markers of terminal differentiation. Notably, the amelioration of the aged phenotype mediated by loss of DJ-1 was only observed in aged mice and not young mice, and was largely found, via BM chimeras, to be a cell-intrinsic effect of DJ-1 KO. Ostensibly, these data appear to support the contention that DJ-1 plays a role in mediating age-related effects on CD4 and CD8 T cells, with removal of DJ-1 restoring, at least phenotypically, these cells to a younger status.
It is concerning to me that there is no analysis of sensitivity of these cells to stimulation, either in aged or young mice. Given that age-related senescence and/or exhaustion in memory phenotype cells (both antigen-experienced memory cells and inexperienced virtual memory cells) is thought to be due to prolonged or chronic stimulation, it seems possible that the DJ-1 KO cells are refractory to stimulation either through the TCR or cytokine stimulation, which may indirectly prevent an ageing phenotype. This appears to be supported by the transcriptional analysis of Tconv CD4+ cells (Extended data Fig 3I), the mixed bone marrow chimera experiments showing reduced Tem cells, and also by reduced expression of KLRG-1, which, it should be noted is not so much known as an immunoaging marker as a marker of activation and terminal differentiation. Also, the overall reduction in Ki67+ cells also suggests a loss of sensitivity to either tonic TCR stimulation or homeostatic cytokine stimulation. The authors should stimulate young WT and DJ-1 KO cells through the TCR (anti-CD3 rather than PMA/ionomycin) and via cytokines to investigate this. Similarly, if the authors are suggesting that loss of DJ-1 ameliorates an ageing phenotype, this should be analysed functionally, by stimulating KO and WT cells and assessing proliferation, in addition to the phenotypic analysis.
It seems an obvious T cell population to analyse in the context of ageing is the virtual memory population (TVM cells; CD44hi CD49dlo) which have been shown to significantly increase proportionally with age and are contained within the conventional TCM population (CD44hi CD62Lhi). Moreover, of the antigen-inexperienced population, they contain the vast majority of the age-related proliferative T cell dysfunction and express multiple markers of senescence (Quinn et al, Cell Rep 2018). It would be interesting to know the impact of the DJ-1 KO on the Tvm population.
The mixed BM chimera data (ext data fig 4) is unclear to me. It seems the mixed BM chimeras were used to try to distinguish whether the changes observed in the global DJ-1 knockouts were intrinsic or extrinsic to the haematopoietic compartment. However, firstly, changes were observed in the young BM chimeras that were not observed in the young global knockouts. It is difficult to understand how this can be interpreted as suggesting a "hematopoietic-intrinsic role for regulating ... the expression of immunoaging related markers" given this was done in young mice. Again, generally speaking and especially in young mice, KLRG-1 and PD-1 are not immunoaging-related markers; they are markers of activation and terminal differentiation. I presume that this statement "...DJ-1 exhibits an aging-BM-independent.... role ..." is because shifts from WT were observed whether or not the DJ-1 KO BM used was from young or old mice. But it is difficult to know what this means given that such changes were not observed in young global DJ-1 KO mice.
The transfer of aged BM assesses the impact of DJ-1 only in cells that are generated de novo in old age and ignores the likelihood that much of the T cell ageing phenotype typically observed is driven by prolonged T cell survival and exposure to stimuli over an extended period of time. This is particularly relevant if loss of DJ-1 makes cells more refractory to stimulation.
It is not clear to me why the mixed BM chimera results with respect to the Tn cells in particular were interpreted as either a haematopoietic-extrinsic or aging dependent phenomena. It is already clear from Figure 2b that the changes in Tn in DJ-1 KO mice are aging related. So given the mixed BM chimeras were young, one would not have expected a shift in relative percentages of WT and DJ-1 KO Tn cells. In light of this, I don't understand why the "haematopoietic-extrinsic" interpretation is being suggested.
The study makes an interesting observation regarding a level of maintenance of mitochondrial mass and membrane potential in aged DJ-1 KO mice that ostensibly aligns with the other markers of aging. However, this appears to be at odds with observations that mitochondrial mass increases in T cells with age (Quinn et al, Nat Commun, 2020). This should be discussed and relevant literature cited.
In summary, the study clearly shows a phenotype of DJ-1 knockout, however it should investigate whether the amelioration of the ageing phenotype by DJ-1 KO is manifest functionally as well as phenotypically, should investigate the impact on TVM cells, and should discern whether it is secondary to an overall reduced sensitivity to stimulation by TCR and/or cytokines.

Minor Points
The analysis of Ki67 staining in homeostatic conditions (Fig 2) doesn't address the question of T cell activation vs cell exhaustion, as stated. Homeostatic proliferation is not a reflection of activation. This analysis investigates whether homeostatic proliferation might be responsible for elevated activation/exhaustion markers. This should be reworded. Note that the Britanova et al, 2014 paper cited does not analyse TCR repertoire of naïve cells, but rather total cells from naïve individuals. Thus, the reference does not support the statement "During aging, the TCR repertoire diversity decreases in naïve T cells." and should be removed. In the submitted manuscript 'DJ-1 depletion slows down immunoaging in T-cell compartments', Zeng and colleagues report on the implication of loss of DJ-1 on the T lymphocyte compartment during aging in mouse and human. DJ-1 deficiency is correlated with a reduction in several signs of immunoaging related to T cells, with respect to prevalence of certain subpopulations to the expression of immunosenescence marker.
1. The authors need to make sure that it is clear in any case (esp. in the abstract, introduction and discussion/summary paragraph), that they refer to immunoaging in the T lymphocyte compartment, as the effect on myeloid cells has not been studied.
2. Please note that 'gender' refers to your own perceived sexual identity. The authors should replace gender in all incidences with 'sex'. 3. The authors use the term 'delay' to describe the found phenotypes related to immunoaging. Can this be specified how long this is? Otherwise, I would suggest to write about the 'diminished signs of immunoaging'. The study by Zeng et al describes as possible role of DJ-1 in regulating the aging of the immune system, by claiming that DJ-1 depletion slows down the aging of T cells compartment of adaptive immunity. The manuscript is interesting and does suggest a possible new role of DJ-1, but in my opinion, in the actual form it fails to prove a functional role of DJ-1 in immunoaging, as it does not provide any real insight on the mechanisms that link DJ-1 KO to slow down immunosenescence. In some cases, the rationale of the proposed experiments is not completely clear Major points.
Thera are four major flaws in this manuscript: -I could not find the "classic" immunophenotypic characterization of patients' CD8+ T cells, i.e. Tn/tcm/Tem/Temra, while it is shown for CD4+ T cells in supplementary data. The authors should show this figure. Is there any difference in the proportion of T cell subsets between the proband, the sibling and age and sex-matched controls?
Reply: Thanks for pointing out this critical issue. We now provided the FACS plots for the expression of CD45RO and CCR7 as well as that of CD45RO and CD27 in Figure 1a, b of the revised version. As shown in the figure, according to those classic markers, the proband vs the two siblings had indeed much more naïve CD8 T cells, but fewer TEM CD8 cells, which is clearly in line with what we observed in aged DJ-KO mice and other patient's data. We also noticed that the proband had much fewer CD8 TEMRA (we referred to CD45RO-CCR7-CD27-) and more TCM cells. Now, the data should be more complete and even more convincing. We also added one more sentence in line 84-88 and page 3 to describe these results.
-The observations on CD8+ T cells are phenotypic but do not demonstrate an actual reduced senescence of T cells. One cannot exclude that these cells are phenotypically "younger" but functionally old. Thus, a functional test is needed on patients' T cells to prove that they are actually less senescent, for example by stimulating T cells in vitro with aCD3/aCD28 followed by division rate quantification via CFSE labeling and analysis of T cell division in T cells subsets. -I do not understand the rationale below the choice of performing BM transplantation. As well established in the past, one of the most important causes of T cell compartment senescence is thymus involution, whose progressive shrinking does not allow proper development of new naïve T cells. Have the authors monitor parameters related to thymic output, such as CD31 expression or sjTREC+ T cells in aged mice? Without this element, it is hard to understand if the effect of DJ-1 is a general effect on the immune system or a specific effect on T cells.

Reply: We now analyzed CD31 expression in both CD4 and CD8 T cells. CD31, as a marker of recent thymic emigrant cells, is fully established for CD4 T cells. As expected, the CD31 expression level was lower in aged vs. younger mice, no matter which genotype group.
However, no matter from young and aged mice, we cannot observe a clear difference in CD31 expression among CD4 T cells between DJ-1 KO and WT mice. Therefore, the higher proportion of naïve T cells was not simply attributable to a higher thymic outcome in aged DJ-1 KO mice. Minor points -I strongly suggest not to overlap the use of exhausted and senescent throughout the manuscript. Although overlapping, the two concepts are clearly different, as well described by Xu and Larbi (2017), and sliding from one to the other does not help the comprehension of the message.

It has been recently shown that not only naïve, but also central memory T cells expressed CD31 among CD8 T cells (Newman
Reply: Thanks for this constructive suggestion. We fully agree with the reviewer that exhaustion and senescence are two different concepts and should not have been exchanged or overlapped in using these two terms. In fact, this is one of the essential reasons why we use "Immmunoaging", rather than "immunosenescence" in the title because the former includes both immune aspects happening during the aging process. In any case, we now removed "immunosenescence" in several places, such as the first sentence of the main text, to avoid giving the wrong impression that we have been essentially discussing immunosencescence. We also now cited the paper from Xu and Larbi (2017) to emphasize this aspect. Throughout the manuscript, we now consistently made all the statements about "immunoaging", but not "immunosenescence". In this way, our statements should be more precise.
-Since I do not see the presence of CD3 Ab in the list of Abs used for analysing human T cells, I think it is necessary to show the gating strategy for the immunophenotype. -There are some typos in the text. Please be consistent in how molecules are named in the text (for example IFN-g, not IFNg Reply: We now systemically corrected this type of nomenclature issues throughout the text.
For example, we now consistently use IFN-γ, rather than anything else. We now use IL-2, rather than IL2 and so on.
-I understand that we are in COVID-19 pandemic era, but I think it is superfluous to cite COVID-19 in a paper that has nothing to do with it. Referee #2: The study begins with an index case containing a homozygous DJ-1 mutation that has driven early Reply: Thanks for your detailed explanation on your concern. We now revised that sections and discussed several aspects in the text (line 188-195, page 5-6) to make it clearer. We also briefly addressed and discussed some essential points here.  The transfer of aged BM assesses the impact of DJ-1 only in cells that are generated de novo in old age and ignores the likelihood that much of the T cell ageing phenotype typically observed is driven by prolonged T cell survival and exposure to stimuli over an extended period of time. This is particularly relevant if loss of DJ-1 makes cells more refractory to stimulation.

Reply: We fully agree with the reviewer that the T-cell aging phenotype is very much driven by prolonged T cell survival and stimulation over an extended period of time. As we replied to the comments above, we now particularly stimulated purified naïve CD8 T cells with different doses of CD3 ab. Indeed, we observed that DJ-1 KO naïve CD8 T cells from young
mice were more refractory to TCR stimulation. The compromised (but not fully lost) sensitivity to TCR stimulation in DJ-1 KO CD8 Tn over the long-lasting aging process might eventually contribute to the reduced immunoaging phenotypes (enhanced frequency of CD8

Tn but decreased fraction of CD8 Tem) we observed in this work.
In line with what the reviewer speculated, in fact, we only observed enhanced frequency of

This data clearly indicates that the non-hematopoietic cells (as part of the stimulation resource during the aging process) might play an important role in the observed reduced immunoaging phenotype. During the revision process, we have now performed TCR sensitivity experiments, which again indicates the critical role of prolonged T-cell activation
in DJ-1-mediated immunoaging phenotypes.
Furthermore, as we already discussed in the text, ethically speaking, we cannot use aged mice as recipients (due to the harmful lethal irradiation procedure) and therefore cannot directly address whether the aging micro-environments regulates the immunoaging process in vivo. Since now we performed TCR sensitivity experiments, it can already at least address the related concern in vitro. The study makes an interesting observation regarding a level of maintenance of mitochondrial mass and membrane potential in aged DJ-1 KO mice that ostensibly aligns with the other markers of aging. However, this appears to be at odds with observations that mitochondrial mass increases in T cells with age (Quinn et al, Nat Commun, 2020). This should be discussed and relevant literature cited. In summary, the study clearly shows a phenotype of DJ-1 knockout, however it should investigate whether the amelioration of the ageing phenotype by DJ-1 KO is manifest functionally as well as phenotypically, should investigate the impact on TVM cells, and should discern whether it is secondary to an overall reduced sensitivity to stimulation by TCR and/or cytokines.

It is not
Reply: we fully agree with the reviewer that we need to investigate the aging phenotype of

DJ-1 KO T cells functionally and should investigate the effect on TVM cells. We have now analyzed TVM frequency in both aged and young mice. In line with the notion of reduced immunoaging phenotype in DJ-1 KO mice, we indeed observed reduced frequency in CD8
TVM, from aged DJ-1 KO vs WT mice (added to Fig. 2i,j). But there was no difference in young DJ-1 KO and WT mice.

Furthermore, we also stimulated CD8 Tn isolated from aged mice with different doses of CD3
ab and showed an encouraging 'younger' phenotype at the functional levels, at least in terms of proliferation and activation (new Fig. 4 generated during revision). From young mice, DJ- Fig 4).

Minor Points
The analysis of Ki67 staining in homeostatic conditions (Fig 2) doesn't address the question of T cell activation vs cell exhaustion, as stated. Homeostatic proliferation is not a reflection of activation. This analysis investigates whether homeostatic proliferation might be responsible for elevated activation/exhaustion markers. This should be reworded. Note that the Britanova et al, 2014 paper cited does not analyse TCR repertoire of naïve cells, but rather total cells from naïve individuals. Thus, the reference does not support the statement "During aging, the TCR repertoire diversity decreases in naïve T cells." and should be removed. All in all, this is a great example of combining clinical findings with genetic model systems for further validation with significant interest for the general readers interested in aging. DJ-1 had not been put into context of immunoaging. The manuscript is well-written and the results are convincingly reported. I recommend this manuscript to be published after revising some minor points: Reply: Thanks for the overall appreciation on our work.

Minor comments
1. The authors need to make sure that it is clear in any case (esp. in the abstract, introduction and discussion/summary paragraph), that they refer to immunoaging in the T lymphocyte compartment, as the effect on myeloid cells has not been studied.
Reply: Thanks for stressing this point. Yes, to be precise, we did not mention anywhere the

myeloid cells and we now checked the abstraction, main text (Introduction, Results and
Discussion part) and even figure legends to make sure that we only referred to Immunoaging in T cells.
2. Please note that 'gender' refers to your own perceived sexual identity. The authors should replace gender in all incidences with 'sex'.
Reply: Thanks for pointing out this mistake. We now systematically replaced all "gender" with the word "sex".
3. The authors use the term 'delay' to describe the found phenotypes related to immunoaging. Can this be specified how long this is? Otherwise, I would suggest to write about the 'diminished signs of immunoaging'.
Reply: Thanks for mentioning this aspect. We cannot specify how long the phenotype was delayed. Therefore, we now rephrased everything to either "reduced immunoaging" or "diminished signs of immunoaging" or "diminished immunoaging phenotypes" or "diminished immunoaging features" as the reviewer suggested. This correction has been done even in title and abstract. Reply: Yes, we now corrected the typo "patent" to "patient".
2nd Dec 2021 1st Revision -Editorial Decision Dear Dr. Hefeng, Thank you for submitting your revised manuscript. It has now been seen by two of the original referees.
I apologize for the delay in getting back to you, it took longer than anticipated to receive the referee reports.
Referees acknowledge that the revision significantly improved the manuscript. However, referee #2 has significant remaining concerns. I have discussed these concerns with referee #1. Referee #1 partially agrees with these concerns, but following the discussion, we concluded that the concerns do not invalidate the main message of the manuscript. However, textual revisions are required for addressing these concerns.
In particular, referee #2 finds that 45 week old mice are not old enough to be considered as old mice (points 1 and 2). I have discussed this concern further with referee #1, who agrees with the concern. However, referee #1 finds that mice at this age already start showing the decline of naive T cells and the increase of memory cells. Therefore, please refer to these mice with their age (e.g. 45 week old mice), instead of calling them old mice, throughout the figures and the text. To address the 3rd concern of referee #2, please also present the absolute numbers of Tvm cells. As per the 4th concern of referee #2, referee #1 agrees that young DJ-1 knockout mice exhibit increased CD8 T cell sensitivity, but he/she also finds that this difference increases in old (middle-aged) animals, which suggests that there is an age-dependent effect, it is not a mere effect on sensitivity that is preserved. However, please make sure to discuss and acknowledge that the sensitivity difference observed in already in the young mice, which increases in an age-dependent manner. Please mark the changes in the text.
Furthermore, I need you to address the editorial points below before I can accept the manuscript.
• For technical reasons, we can only accommodate 5 keywords, and there are currently 6. Please remove one of the keywords. • We note that Figures EV1 i-k are currently not called out in the text. • There are callouts to Appendix Fig S1 which need correcting/deleting. • Papers published in EMBO Reports include a 'synopsis' and 'bullet points' to further enhance discoverability. Both are displayed on the html version of the paper and are freely accessible to all readers. The synopsis includes a short standfirst summarizing the study in 1 or 2 sentences (max 35 words) that summarize the paper and are provided by the authors and streamlined by the handling editor. I would therefore ask you to include your synopsis blurb and 3-5 bullet points listing the key experimental findings. • In addition, please provide an image for the synopsis. This image should provide a rapid overview of the question addressed in the study but still needs to be kept fairly modest since the image size cannot exceed 550x400 pixels.
Thank you again for giving us to consider your manuscript for EMBO Reports, I look forward to your minor revision. In the previous review round, my main observations regarded the fact that most observations on the effects of DJ-1 on T cell aging were phenotypic, rather than functional, particularly as far as mitochondrial functionality is concerned. The authors have fully addressed my main concerns; the new data concerning immunometabolic properties of T cells in the DJ-1 mouse model during aging are properly presented and discussed.
I have no further comments or requests.
Referee #2: The authors have done a number of the suggested experiments and provided explanations for existing data. I accept that there is a real consequence of loss of DJ-1 especially as it relates to sensitivity of the T cells to stimulation. However, I do not feel that all issues have been adequately addressed, as outlined below.
-Firstly, it has become clear to me that the 'aged' mice are only 45 weeks old. This is not the widely accepted age range of mice considered to reflect elderly humans. This link defines that ageing in mice is considered to occur from 18-24 months of age (https://www.jax.org/news-and-insights/jax-blog/2017/november/when-are-mice-considered-old#). Certainly our own experience suggests that classical hallmarks of immune aging do not occur until at least 15 months of age. -For this reason, I do not accept that mice that are 20 weeks of age could be considered to be partly aged and find this to be a complicated explanation for the BM chimera data.
-While the authors have quantitated the % of Tvm cells and shown that they do not increase, it is the markers of senescence that are of particular interest. In WT mice, the % of Tvm cells only increases with age because the Tn cells decrease; the # of Tvm cells remains stable. Generally, there are only measures of percentages stated here, rather than absolute numbers which would be more telling.
-The new data showing TCR responsiveness essentially shows reduced sensitivity in DJ-1 KO cells in younger mice and that same sensitivity is maintained with age. Thus, it seems clear that DJ-1 plays a role primarily in sensitivity to stimulation rather than directly in T cell aging per se. This explains a great deal of the data without needing to refer to an 'aging phenotype'. For example, it makes sense that markers of activation such as PD1, Eomes, LAG3 etc would be increased on WT cells that are more receptive to stimulation, that there would be a larger proportion of true naïve T cells, increased clonal diversity, reduced Ki67 etc. This is all relative. There is very little in the way of a definitive measure of 'senescence' or an 'aging' phenotype per se. In the previous review round, my main observations regarded the fact that most observations on the effects of DJ-1 on T cell aging were phenotypic, rather than functional, particularly as far as mitochondrial functionality is concerned. The authors have fully addressed my main concerns; the new data concerning immunometabolic properties of T cells in the DJ-1 mouse model during aging are properly presented and discussed.
I have no further comments or requests. Reply: Thanks for appreciating our work and accepting our work.

Referee #2:
The authors have done a number of the suggested experiments and provided explanations for existing data. I accept that there is a real consequence of loss of DJ-1 especially as it relates to sensitivity of the T cells to stimulation. However, I do not feel that all issues have been adequately addressed, as outlined below.
-Firstly, it has become clear to me that the 'aged' mice are only 45 weeks old. This is not the widely accepted age range of mice considered to reflect elderly humans. This link defines that ageing in mice is considered to occur from 18-24 months of age (https://www.jax.org/news-andinsights/jax-blog/2017/november/when-are-mice-considered-old#). Certainly our own experience suggests that classical hallmarks of immune aging do not occur until at least 15 months of age.
Reply: Thanks for highlighting this aspect. To address the concern raised by the reviewer, we now systematically rephrased "aged mice" as "45-wk-old mice", or specified the age of the old mice if the used mice were much older than 45 weeks in all the main and Expanded View Figures and manuscript text. In some places, we also mentioned the term "middle-aged" for discussion. In fact, when checking the precise age information, we noticed that some of the experiments (e.g., the Tvm analysis) performed during the last revision were based on the mice slightly older than 60 weeks (around 14-month old). Due to the long-lasting nature of the project, we have almost no chances to strictly to only work with mice within a very narrow window of age. The effect of DJ-1 on the frequency of CD8 T-cell subsets in 60-wk-old mice has already been shown in main Figure 2 (i-l) even in the last revision. In the updated Figure EV2 (c-g) of the current revision, where we displayed the cell numbers of different CD8 subsets, the same effect of DJ-1 on immunoaging phenotypes has been kept. To facilitate the reviewing process, we directly copied the updated Figure EV2 below.
Last but not least, as noticed by another reviewer as mentioned in the decision letter, the immunoaging hallmarks we observed here could already happen in 45-wk-old mice in their laboratories. The difference in the age starting to show immunoaging hallmarks might be attributable to hygiene degree in different animal In short, our observations, although the majority of which were made in 45-wk-old mice, are still valid and well support our major statements.
-For this reason, I do not accept that mice that are 20 weeks of age could be considered to be partly aged and find this to be a complicated explanation for the BM chimera data.
Reply: Thanks for pointing out this aspect. We agree with the reviewer that this explanation is not ideal. We now removed the related sentences in the text. We believe that the lymphopenia-induced hyperactive reconstitution and the accelerated development of the immune system might be already a sufficient and plausible explanation for the BM chimera data.
-While the authors have quantitated the % of Tvm cells and shown that they do not increase, it is the markers of senescence that are of particular interest. In WT mice, the % of Tvm cells only increases with age because the Tn cells decrease; the # of Tvm cells remains stable. Generally, there are only measures of percentages stated here, rather than absolute numbers which would be more telling.
Reply: Thanks for suggesting this. We now checked the original datasets and calculated the absolute number of CD8 Tn, Tvm and Tmem cells within the same amount (2e4) of acquired living CD3 T cells from young or 60-wk-old mice. We also directly copied the updated Figure EV 2 in the response letter below. Clearly, even in terms of absolute number, DJ-1 depletion also reduced T-cell immunoaging signs in 60-wk-old mice. With our experimental setting, we were unable to precisely estimate the cell number of CD8 Tvm in total splenocytes. Different from many others, we stopped the flow cytometry acquisition, when the same number (20K) of living CD3 T cells was achieved, to make sure that the flow cytometry plots are really comparable.
In any case, the absolute numbers of CD8 subsets within the same amount of living CD3 T cells are already informative enough. Considering there were no significant changes in both CD3 frequency and total splenocytes between 60-wk-old Dj-1 KO and WT mice, the estimated absolute numbers of CD8 subsets in total splenocytes should retain a similar pattern as the absolute numbers of CD8 subsets within the same amount of total CD3 T cells. We have also now described the related results in line 161-174, page 5.
-The new data showing TCR responsiveness essentially shows reduced sensitivity in DJ-1 KO cells in younger mice and that same sensitivity is maintained with age. Thus, it seems clear that DJ-1 plays a role primarily in sensitivity to stimulation rather than directly in T cell aging per se.
This explains a great deal of the data without needing to refer to an 'aging phenotype'. For example, it makes sense that markers of activation such as PD1, Eomes, LAG3 etc would be increased on WT cells that are more receptive to stimulation, that there would be a larger proportion of true naïve T cells, increased clonal diversity, reduced Ki67 etc. This is all relative. There is very little in the way of a definitive measure of 'senescence' or an 'aging' phenotype per se.
Reply: We agree with the reviewer that the new TCR sensitivity data essentially showed that the TCR sensitivity of DJ-1 KO CD8 Tn was quite preserved between the young and older mice. We also agree that the TCR sensitivity was compromised already at a younger age. Nevertheless, the preserved TCR sensitivity alone cannot fully explain why we only observed all the immune cellular phenotypes in older (at least 45-wk-old) mice, but not in younger mice. In fact, as shown in Figure 4, the difference in TCR sensitivity between DJ-1 KO and WT mice that already started from a young age increased in an aging-dependent manner. In another words, during natural aging, TCR sensitivity of CD8 Tn decreased dramatically in WT mice, while that in DJ-1 KO CD8 Tn was declined not as dramatically as in WT mice. Of note, to more logically discuss the aspects raised by the reviewer, we now swapped the position of subpanels of TCR sensitivity data between young and older mice in Figure 4.
Furthermore, if we consider Figure 2b, c and many other cellular data presented in this work, a similar observation reoccurred throughout the manuscript. That is, the immunoaging markers showed a dramatic increase (or decrease) in WT mice, but that showed a much milder change in DJ-1 KO mice during the aging process. The differences in the changing slopes over time of various immunoaging markers between DJ-1 KO and WT mice together contributed to a relatively reduced immunoaging phenotype in DJ-1 KO mice.
Importantly, we would like to stress again, almost no cellular phenotypes (e.g., frequency of naïve and memory T cells etc.) appeared in young mice, clearly indicating an aging-dependent phenotype.
We have also now discussed the better preserved TCR sensitivity in DJ-1 KO mice and also the fact that the difference in TCR sensitivity between DJ-1 KO and WT mice already starting from a young age increases in an aging-dependent manner in line 303-307, page 9 and line 395-402, page 11.
Minor points:--Why are CD27+CD28+ CD8 T cells defined as 'non-senescent'? They are the definition of senescence in CD4+ T cell populations, for example in many studies by Akbar et al.
Reply: Thanks for noticing this potential aspect. We just checked the papers from Akbar and colleagues again.
We are sure that CD27+CD28+ CD8 T cells are non-senescent T cells. For instance, in the recent paper by Therefore, we are confident that CD27+CD28+ CD8 T cells we referred here should be called "non-senescent" T cells and might be even called CD8 Tn if we are not conservative enough.
Reply: We agree with the reviewer that the staining for PD-1, Eomes, CD57 and T-bet in human samples looks not so great. While we cannot change the staining quality anymore, we tried to improve the visualization of those panels of Figure 1 by changing the axis scales of flow cytometry plots (e.g., changing from 10 5 to 10 4 to zoom in the populations in some cases). But we cannot get better ones for CD57. In the current revision, we also replaced the previous CD27 VS CD28 plots with better ones. In any case, even with the previous version, they all can deliver a clear and consistent message that the PD index patient (P2) had a much smaller fraction of cells expressing those markers than the two siblings. In combination with all the other results, we are confident that the flow cytometric data about those markers are reliable. To simplify the reviewing procedure, we also copied the updated part of Figure 1 below. --At the end of this email I include important information about how to proceed. Please ensure that you take the time to read the information and complete and return the necessary forms to allow us to publish your manuscript as quickly as possible.
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Journal Submitted to: EMBO Reports
Corresponding Author Name: Feng Q. Hefeng

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
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definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).

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Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
For each subfigure related to mice, in most cases, at least five mice were chosen per genotype group at the given age and have been repeated at least three times in different cohorts. For the TVM and CD31 analysis, we chose at least three mice per genotype group at the given age. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
We chose at least 5 mice per genotype and age group (the same sex) for each analysis and each analysis has been repeated at least three times.

NA.
For the immunoaging phenotype analysis in mice, the WT or KO littermates were always maintained in the same cages. For the bone marrow transplanation experiments, the CD45.1 and CD45.2 mixture were transferred into the same recipients and therefore we tried our best to minimize the potential bias in our projects.

Yes
All the detailed information of the used antibodies and the key reagents have been provided in the Appendix. We added here again. We only used antibodies commercially available, which have been routinely validated by the providers and often by other literatures. Table S1. List of mouserelated antibodies used in this work. Antibody Clone Company Catalogue number Dilution factor and application* CD16/CD32 2. The operators were blind to the genetic information of the three human participants before the analysis. The operators were also blind to the genetic group of mice before finishing the flow cytometry analysis. added in the Materials and Methods section under the subheading "Animals"

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.