Redox regulation of PTPN22 affects the severity of T-cell-dependent autoimmune inflammation

  1. Jaime James
  2. Yifei Chen
  3. Clara M Hernandez
  4. Florian Forster
  5. Markus Dagnell
  6. Qing Cheng
  7. Amir A Saei
  8. Hassan Gharibi
  9. Gonzalo Fernandez Lahore
  10. Annika Åstrand
  11. Rajneesh Malhotra
  12. Bernard Malissen
  13. Roman A Zubarev
  14. Elias SJ Arnér
  15. Rikard Holmdahl

  • Curated by eLife

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    Evaluation summary:

    This paper documents a novel aspect of how T cell activation is regulated by the PTPN22 phosphatase, namely reversible oxidation which transiently reduces the activity of PTPN22 to allow the T cell antigen receptor to drive a strong activation signal. This compelling work adds to our understanding of how an immune response is initiated and provides new insights that could be exploited for the development of new drugs to treat immune-mediated diseases.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor (TCR) signaling. As with all protein tyrosine phosphatases, the activity of PTPN22 is redox regulated, but if or how such regulation can modulate inflammatory pathways in vivo is not known. To determine this, we created a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 (C129S), a residue proposed to alter the redox regulatory properties of PTPN22 by forming a disulfide with the catalytic C227 residue. The C129S mutant mouse showed a stronger T-cell-dependent inflammatory response and development of T-cell-dependent autoimmune arthritis due to enhanced TCR signaling and activation of T cells, an effect neutralized by a mutation in Ncf1, a component of the NOX2 complex. Activity assays with purified proteins suggest that the functional results can be explained by an increased sensitivity to oxidation of the C129S mutated PTPN22 protein. We also observed that the disulfide of native PTPN22 can be directly reduced by the thioredoxin system, while the C129S mutant lacking this disulfide was less amenable to reductive reactivation. In conclusion, we show that PTPN22 functionally interacts with Ncf1 and is regulated by oxidation via the noncatalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T-cell-dependent autoimmunity.

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  1. Version published to 10.7554/elife.74549 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This is a very solid and exciting study.

    We thank the reviewer for finding our study to be very solid and exciting.

    I have several suggestions, comments and questions:

    1. The authors focused on examining the role of C129 as a regulator of PTPN22 redox sensitivity based on a published crystal structure of the catalytic domain. It would be great if they could demonstrate the existence of the disulfide bond between C129 and C227 also experimentally (in T cells).

    As we understand it, it is requested that the disulfide bond between C227 and C129, as previously suggested by Tsai et al. (2009) (1) with pure protein, should be documented to actually occur in the activated T cells. We fully agree that this would improve the study and we have therefore made several attempts to demonstrate this oxidation, or the oxidation state of the active site Cys residue in PTPN22 in situ. However, as we had also expected, it has proven to be technically very challenging. Nevertheless, as the functional consequence of the PTPN22 oxidation and the effect of the C129S mutation is clearly documented in the mouse, using in vivo experiments, we still think it is valid to conclude that the reversible oxidation state of PTPN22 as well as the involvement of the Cys129 residue regulates the function of PTPN22 in vivo, which is the main conclusion of our study.

    1. To this end, there are other cysteine residues in the vicinity of C227 such as the C231 that might be involved in the redox regulation PTPN22. The authors should at least discuss the their possible involvement.

    It is correct that Tsai et al. (2009) (1) found that mutating C231 to serine dramatically reduced phosphatase activity, thus suggesting its importance in catalysis. Reactivation assays showed higher reactivation rates for C231S mutants, and they suggested that C231 suppresses reactivation in a reducing environment by competing with C227 for reduction in the catalytic pocket. Therefore, C231 could also be a target for negative regulation of PTPN22. However, our project was from the start limited to the intention of studying whether PTPN22 could be shown to be redox regulated in vivo through modification of key cysteine residues, and the aim has not been to give the full picture of how the molecule is regulated. We have now extended this point in the discussion in the paper.

    1. How is mutation of C227 affecting T cell function? Are the effects similar with those of C129S?

    This would be interesting but to analyze if also the cysteine at 227 is regulating the T cell activation by creating another transgenic C227S mouse is outside the scope of the study. As said above and clearly described in the study, we have focused on the redox-mediated effects through C129 and hope that the reviewer can agree with us that this rather focused study is solid and fully sufficient for publication on its own merits.

    1. Although the in vitro evaluation of the PTPN22 activity is of highest quality, it would be good to demonstrate that C227 redox status is modified under physiological conditions. 25-100 µM H2O2 is a high concentration that might not be reached within a cell and might be lethal for T cells.

    See response to point 1.

    1. C129 seems not to be mutated in patients with autoimmunity but is an excellent tool to test the importance of C227 redox regulation and the findings of this study suggest that its over-oxidation will support autoimmune responses. When considering the clinical relevance of the study, a drug that will protect the oxidation of the catalytic cysteine and/or stabilize the disulfide bond would have beneficial effects. The authors could test such pharmacological modulators in isolated T cells.

    Indeed, such modulators would be very interesting to test; however, developing such drugs can hardly be demanded to be within the scope of this study. We have however included a statement on this topic in the Discussion of the manuscript.

    1. The authors discuss that NOX2-derived ROS most likely originate from antigen presenting cells. I fully agree with this discussion. However, some studies have proposed that NOX2 plays an important role also in T cells, a finding which was not confirmed by other following studies. It would be great if the authors could address this controversial issue in regards to their findings.

    The finding that the ROS that modify PTPN22 in fact come from the interacting APC rather than from the T cell itself we believe is very important. However, we have not made a major point of this as we have shown that aspect before in other studies, and we wanted in the current paper to focus on the take home message that PTPN22 could hereby be shown to be redox regulated in vivo. However, the last word about the source of ROS has not been said. The controversy whether the Ncf1 containing NOX2 complex is functionally expressed in T cells stems from the paper by Jackson et al. in Nat Immunol 2004 (2). We have not been able to reproduce those findings and in addition we have never detected a NOX2 dependent response in pure T cells, which has also been shown in several of our papers. There are certainly many pitfalls, contaminating NOX2 expressing cells, NOX2 containing exosomes and peroxides, and even NOX2 complexes picked up by interactions with antigen presenting cells. However, it is dangerous to completely exclude that Ncf1 could be expressed at minimal levels or to exclude that functional NOX2 complex can indeed be formed in T cells, and we all know that minute levels of any peroxide as produced by cells could have an impact on cellular functions. But, based on the present knowledge we conclude that T cells do not functionally express Ncf1-containing NOX2 complexes. We have now added two references to enlighten this point, (3, 4; refs. 38 & 39 in the manuscript).

    1. Fig. 1: Is the addition of bicarbonate affecting the pH and thus the activity of PTPN22?

    No, we believe that addition of bicarbonate is not acting by an altered pH but is instead required for formation of peroxymonocarbonate when reacting with H2O2, which is subsequently the molecular species that bypasses the cellular antioxidant systems in order to oxidize the active site Cys residues of target PTPs. This was shown by us in an earlier publication (Dagnell et al, ref. 11 in the manuscript) (5) and a sentence has now been added in the Discussion to further emphasize this point.

    1. The H2O2 concentration dependence of PTPN22_C129S should also be shown as for WT (see Fig. 1B)

    We agree with the reviewer that titration of the mutant with additional H2O2 concentrations could potentially have been done, but we thought that the comparison of WT and C129S enzyme side-by-side using either 0 µM, 25 µM or 50 µM as in Fig. 1D was a sufficient comparison in H2O2 sensitivity. Unfortunately, we do not have the possibility to analyze more purified C129S mutant protein at the moment and it would require a major effort to run those additional experiments. We thereby hope that the reviewer would agree with having the data presented as they currently are to be sufficient.

    1. Quantification of the slope based on only 3 measuring points is not accurate (Fig. 1D).

    Each data point in those curves represents the mean ± S.D. derived from duplicate samples ran three different times, with clearly very low standard deviations. Thus, we believe that the data are reliable and that the statistically significant difference when comparing the slopes between WT and the C129S mutant as shown in the figure, should be trustworthy.

    1. The pinna thickness measurements shown in Fig. 3B and C suggest that in NCF1 mice C129S has no effect. However, the thickness in NCF1 mice is already much higher than in WT mice (compare B and C). Does this mean that NOX2-derived ROS are the only factor that affects C227 redox properties?

    The effects of the decreased ROS due to the Ncf1 mutation is likely to have consequences for the functions of many proteins, in different pathways, and not only of PTPN22. The sum effect is that the Ncf1 mutated mice responds stronger than the wild type, which explains the difference. However, the main message here is that if there is no ROS from the NOX2 complex, the effect of the PTPN22 mutation is lost.

    1. The results shown in Fig. 5D could be moved to a supplementary figure.

    We prefer to keep it within Fig 5 as it is more logical in the context or the other parts of this figure. Of course, if there is a space layout problem, we can consider moving it.

    1. The calcium measurements are not convincing and the differences are rather small. The y axis labels show 50K, 100K etc. Are this ratio values? If yes the imaging settings need to be optimized. Why is the mutant labeled as Pep? How is the C129S affecting calcium signaling? These observations need be examined in more detail or maybe calcium is not playing an important role.

    We agree that the differences in calcium measurements are not very large but have nevertheless been repeated several times, and there is a significant difference as shown. The calculation is done on the slope of the curve, which is independent of the absolute values given on the y-axis. We agree that the figure was not properly labeled and have now changed this.

    1. I would suggest a more extensive evaluation of the proteomic data presented in Fig. 6D. The results might be very exciting and can further increase the impact of this study.

    We fully agree with this. We have chosen not to go into details of the results of the proteomic analysis. The data shown confirms our conclusion and we did not plan to identify the downstream targets of the PTPN22 oxidative regulation. Highlighting some of these targets will require biological confirmation, which can be done but must await future work. The full dataset has however been deposited in PRIDE for any reader interested to analyze the results further.

    1. Is 24h BSO treatment not toxic for the T cells (ferroptosis)?

    We have not seen any evidence for toxicity upon the BSO treatment of T cells in vitro, which however has been more thoroughly checked by others. Gringhuis et al (JI, 2000) (6) have shown immunofluorescence staining on T cells 72 hours post BSO treatment with intact cell membranes. Additionally, Carilho et al. (Chem. Cent. J., 2013, 7:150) (7) noted no changes in Jurkat T cell viability after 24 hours at a maximum dose of 100 µM BSO.

    Reviewer #3 (Public Review):

    The manuscript by James, Chen Hernandez et al. reveals a novel function for PTPN22 oxidation in T-Cell activation. The authors used a broad array of methods to demonstrate that PTPN22 is catalytically impaired in addition to being more sensitive to reversible oxidation in vitro. In the characterization process, the authors found that PTPN22 could be directly reduced by Thioredoxin Reductase and that oxidation of PTPN22 oxidation could be easily monitored by the appearance of a faster migrating band in non-reducing gels. Supporting the hypothesis that the catalytic Cysteine forms a disulfide with a backdoor Cysteine (Cys129), the authors found that this C129S mutant is prone to oxidation and cannot be reduced back to its active form by Thioredoxin Reductase. Using a new mouse model in which this key Cysteine of PTPN22 is mutated to a Serine residue (PTPN22C129S mutant) and can presumably not form a stabilizing redox intermediate between the catalytic Cys residue and this backdoor Cys (C227-C129), the authors study how the oxidation prone mutant affects T-Cell activation. The authors find that the C129S mutant mouse showed an increased T-Cell dependent inflammatory response that was dependent on activation of the reactive oxygen species-producing enzyme NOX2. This data adds an interesting redox twist to the function of PTPN22 in T-Cells that contributes to conversation on the protective effects of reactive oxygen species against inflammatory diseases in vivo.

    Strengths:

    The in vitro characterization of the WT and C129S mutant form of PTPN22 is very thorough. Determination of the Km and Kcat highlights the differences between the two enzymes that go beyond redox regulation of the phosphatase. The reduction studies are masterfully done and highlight a novel reduction mechanism that merits to be further studied in cells. Demonstrating that PTPN22C129S is prone to oxidation in vitro is a key and technically challenging result that may be applicable to other members of the PTP family that also form disulfides with a backdoor cysteine. Showing that PTPN22C129S mice (backcrossed to B6Q mice making them susceptible to autoimmune arthritis) displayed higher T cell activation in two models (DTH and GPI), in addition to studies in T cells stimulated with collagen, increased this reviewer's confidence that the PTPN22C129S mouse exhibited T-cell-dependent inflammatory response phenotype similar to the PTPN22 knockout phenotype. Validation of T-cell signaling events in PTPn22C129S T cells were in line with the in vitro characterization of the phosphatase.

    We thank the reviewer very much for the detailed summary of our findings and the appreciative words.

    Weaknesses: Although the paper has many strengths, some important weaknesses need to be addressed by the authors. In particular, the authors need to characterize better their mouse model and determine if PTPN22 is reversibly oxidized following TCR activation. If PTPN22 is oxidized, does it form an intramolecular disulfide between C227 and C129? The proposed model, that PTPN22C129S is more prone to oxidation, also has to be validated in vivo. Although this could be technically challenging in theory, the authors have shown that the migration pattern of the oxidized enzyme is different that of the reduced enzyme. Another major issue is that PTPN22 does not appear to be expressed in CD4+ T cells unless these cells are activated in vitro with anti-CD3/CD28 for 24 hours. This makes acute CD3-stimulation of CD4+ T cells studies - such as the measurement of acute calcium influx in Fig. 5E - very difficult to interpret. Perhaps the authors should explain why acute signal transduction studies in Figure 6 were performed in lymph node cells. If the reason is that PTPN22 (WT and C129S mutant) expression is higher, the authors should provide immunoblots for PTPN22 in these cells. Since the PTPN22C129S mouse model has not been sufficiently validated, the claims of the authors are unfortunately weakened and the underlying molecular mechanisms do not completely support their conclusions. However, given the clear in vitro work provided in figures 1 and 2, it is this Reviewer's opinion that the authors can address the issues related to the oxidation status of PTPN22 and of PTPN22C129S in vivo, support their claims, and make a significant contribution to the field.

    We again thank the reviewer for the detailed summary of our findings and for the suggestions. With regards to the in vivo oxidation status of PTPN22, please see the discussion above.

    1. Tsai SJ, Sen U, Zhao L, Greenleaf WB, Dasgupta J, Fiorillo E, et al. Crystal structure of the human lymphoid tyrosine phosphatase catalytic domain: insights into redox regulation. Biochemistry. 2009;48(22):4838-45.
    2. Jackson SH, Devadas S, Kwon J, Pinto LA, Williams MS. T cells express a phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation. Nat Immunol. 2004;5(8):818-27.
    3. Gelderman KA, Hultqvist M, Holmberg J, Olofsson P, Holmdahl R. T cell surface redox levels determine T cell reactivity and arthritis susceptibility. Proc Natl Acad Sci U S A. 2006;103(34):12831-6.
    4. Gelderman KA, Hultqvist M, Pizzolla A, Zhao M, Nandakumar KS, Mattsson R, et al. Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species. J Clin Invest. 2007;117(10):3020-8.
    5. Dagnell M, Cheng Q, Rizvi SHM, Pace PE, Boivin B, Winterbourn CC, et al. Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades. J Biol Chem. 2019;294(33):12330-8.
    6. Gringhuis SI, Leow A, Papendrecht-Van Der Voort EA, Remans PH, Breedveld FC, Verweij CL. Displacement of linker for activation of T cells from the plasma membrane due to redox balance alterations results in hyporesponsiveness of synovial fluid T lymphocytes in rheumatoid arthritis. J Immunol. 2000;164(4):2170-9.
    7. Carilho Torrao RB, Dias IH, Bennett SJ, Dunston CR, Griffiths HR. Healthy ageing and depletion of intracellular glutathione influences T cell membrane thioredoxin-1 levels and cytokine secretion. Chem Cent J. 2013;7(1):150.
  3. eLife

    Evaluation summary:

    This paper documents a novel aspect of how T cell activation is regulated by the PTPN22 phosphatase, namely reversible oxidation which transiently reduces the activity of PTPN22 to allow the T cell antigen receptor to drive a strong activation signal. This compelling work adds to our understanding of how an immune response is initiated and provides new insights that could be exploited for the development of new drugs to treat immune-mediated diseases.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This is a very solid and exciting study. I have several suggestions, comments and questions:

    The authors focused on examining the role of C129 as a regulator of PTPN22 redox sensitivity based on a published crystal structure of the catalytic domain. It would be great if they could demonstrate the existence of the disulfide bond between C129 and C227 also experimentally (in T cells). To this end, there are other cysteine residues in the vicinity of C227 such as the C231 that might be involved in the redox regulation PTPN22. The authors should at least discuss the their possible involvement.

    How is mutation of C227 affecting T cell function? Are the effects similar with those of C129S?

    Although the in vitro evaluation of the PTPN22 activity is of highest quality, it would be good to demonstrate that C227 redox status is modified under physiological conditions. 25-100 µM H2O2 is a high concentration that might not be reached within a cell and might be lethal for T cells.

    C129 seems not to be mutated in patients with autoimmunity but is an excellent tool to test the importance of C227 redox regulation and the findings of this study suggest that its over-oxidation will support autoimmune responses. When considering the clinical relevance of the study, a drug that will protect the oxidation of the catalytic cysteine and/or stabilize the disulfide bond would have beneficial effects. The authors could test such pharmacological modulators in isolated T cells.

    The authors discuss that NOX2-derived ROS most likely originate from antigen presenting cells. I fully agree with this discussion. However, some studies have proposed that NOX2 plays an important role also in T cells, a finding which was not confirmed by other following studies. It would be great if the authors could address this controversial issue in regards to their findings.

    Fig. 1: Is the addition of bicarbonate affecting the pH and thus the activity of PTPN22?

    The H2O2 concentration dependence of PTPN22_C129S should also be shown as for WT (see Fig. 1B)

    Quantification of the slope based on only 3 measuring points is not accurate (Fig. 1D).

    The pinna thickness measurements shown in Fig. 3B and C suggest that in NCF1 mice C129S has no effect. However, the thickness in NCF1 mice is already much higher than in WT mice (compare B and C). Does this mean that NOX2-derived ROS are the only factor that affects C227 redox properties?

    The results shown in Fig. 5D could be moved to a supplementary figure.

    The calcium measurements are not convincing and the differences are rather small. The y axis labels show 50K, 100K etc. Are this ratio values? If yes the imaging settings need to be optimized. Why is the mutant labeled as Pep? How is the C129S affecting calcium signaling? These observations need be examined in more detail or maybe calcium is not playing an important role.

    I would suggest a more extensive evaluation of the proteomic data presented in Fig. 6D. The results might be very exciting and can further increase the impact of this study. Is 24h BSO treatment not toxic for the T cells (ferroptosis)?

  5. eLife

    Reviewer #2 (Public Review):

    In this paper, the authors examine the acute regulation of the PTPN22 tyrosine phosphatase, an important negative regulator of T cell antigen receptor signaling, but reversible oxidation. PTPN22 has a cysteine residue C129 in close proximity to its catalytic cysteine, C227, allowing the formation of a disulfide bond between them, rather than an irreversible oxidation of C227, under conditions of reactive oxygen species (ROS) production. By mutating C129 to serine, the authors report that PTPN22 function is significantly altered during T cell activation, resulting in increased downstream effects of TCR signaling and worse autoimmune arthritis. Using a loss-of-function mutant of the NOX2 machinery, they provide further evidence that ROS production is key to PTPN22 inactivation during TCR activation.

    The methods used are appropriate, well controlled, and the results clearly presented and interpreted. Experiments range from protein chemistry in vitro to assessment of T cell function in ptpn22-C129S expressing animals in an autoimmune arthritis model.

    The proposed model of PTPN22 regulation fits well into the current understanding of PTP regulation and the role of PTPN22 in TCR signaling. Indeed, the very rapid increase in tyrosine phosphorylation of numerous proteins after TCR ligation is readily explained by not only tyrosine kinase activation, but by a simultaneous and transient inhibition of PTPN22.

  6. eLife

    Reviewer #3 (Public Review):

    The manuscript by James, Chen Hernandez et al. reveals a novel function for PTPN22 oxidation in T-Cell activation. The authors used a broad array of methods to demonstrate that PTPN22 is catalytically impaired in addition to being more sensitive to reversible oxidation in vitro. In the characterization process, the authors found that PTPN22 could be directly reduced by Thioredoxin Reductase and that oxidation of PTPN22 oxidation could be easily monitored by the appearance of a faster migrating band in non-reducing gels. Supporting the hypothesis that the catalytic Cysteine forms a disulfide with a backdoor Cysteine (Cys129), the authors found that this C129S mutant is prone to oxidation and cannot be reduced back to its active form by Thioredoxin Reductase. Using a new mouse model in which this key Cysteine of PTPN22 is mutated to a Serine residue (PTPN22C129S mutant) and can presumably not form a stabilizing redox intermediate between the catalytic Cys residue and this backdoor Cys (C227-C129), the authors study how the oxidation prone mutant affects T-Cell activation. The authors find that the C129S mutant mouse showed an increased T-Cell dependent inflammatory response that was dependent on activation of the reactive oxygen species-producing enzyme NOX2. This data adds an interesting redox twist to the function of PTPN22 in T-Cells that contributes to conversation on the protective effects of reactive oxygen species against inflammatory diseases in vivo.

    Strengths:

    The in vitro characterization of the WT and C129S mutant form of PTPN22 is very thorough. Determination of the Km and Kcat highlights the differences between the two enzymes that go beyond redox regulation of the phosphatase. The reduction studies are masterfully done and highlight a novel reduction mechanism that merits to be further studied in cells. Demonstrating that PTPN22C129S is prone to oxidation in vitro is a key and technically challenging result that may be applicable to other members of the PTP family that also form disulfides with a backdoor cysteine. Showing that PTPN22C129S mice (backcrossed to B6Q mice making them susceptible to autoimmune arthritis) displayed higher T cell activation in two models (DTH and GPI), in addition to studies in T cells stimulated with collagen, increased this reviewer's confidence that the PTPN22C129S mouse exhibited T-cell-dependent inflammatory response phenotype similar to the PTPN22 knockout phenotype. Validation of T-cell signaling events in PTPn22C129S T cells were in line with the in vitro characterization of the phosphatase.

    Weaknesses:

    Although the paper has many strengths, some important weaknesses need to be addressed by the authors. In particular, the authors need to characterize better their mouse model and determine if PTPN22 is reversibly oxidized following TCR activation. If PTPN22 is oxidized, does it form an intramolecular disulfide between C227 and C129? The proposed model, that PTPN22C129S is more prone to oxidation, also has to be validated in vivo. Although this could be technically challenging in theory, the authors have shown that the migration pattern of the oxidized enzyme is different that of the reduced enzyme. Another major issue is that PTPN22 does not appear to be expressed in CD4+ T cells unless these cells are activated in vitro with anti-CD3/CD28 for 24 hours. This makes acute CD3-stimulation of CD4+ T cells studies - such as the measurement of acute calcium influx in Fig. 5E - very difficult to interpret. Perhaps the authors should explain why acute signal transduction studies in Figure 6 were performed in lymph node cells. If the reason is that PTPN22 (WT and C129S mutant) expression is higher, the authors should provide immunoblots for PTPN22 in these cells.

    Since the PTPN22C129S mouse model has not been sufficiently validated, the claims of the authors are unfortunately weakened and the underlying molecular mechanisms do not completely support their conclusions. However, given the clear in vitro work provided in figures 1 and 2, it is this Reviewer's opinion that the authors can address the issues related to the oxidation status of PTPN22 and of PTPN22C129S in vivo, support their claims, and make a significant contribution to the field.

  7. Version published to 10.1101/2021.12.02.470976v1 on bioRxiv