The cytokine receptor DR3 identifies and activates thymic NKT17 cells

  1. Shunqun Luo
  2. Nurcin Liman
  3. Assiatu Crossman
  4. Jung-Hyun Park

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    Evaluation Summary:

    The manuscript by Luo et al. showed that the cytokine receptor DR3 is selectively expressed on thymic NKT17 cells and DR3 ligation leads to the activation of NKT17 cells in the thymus. Overall, The presented experiment are properly executed, controlled and presented. The finding that DR3 acts as a costimulatory molecule for thymic NKT17 cells is interesting. The mechanism and the functional relevance of this finding remain wanting.

    (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 #1 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Invariant natural killer T ( i NKT) cells are thymus-generated T cells with innate-like characteristics and effector function. Several functionally distinct i NKT subsets have been identified, but NKT17 is the only i NKT subset that produces the proinflammatory cytokine IL-17. NKT17 cells are generated in the thymus and then exported into the periphery to populate lymphoid organs and barrier tissues, such as the lung, to provide critical support in host defense. However, the molecular mechanisms that drive the thymic development and subset-specific activation of NKT17 cells remain mostly unknown. Here, we identify the cytokine receptor DR3, a member of the TNF receptor superfamily, being selectively expressed on NKT17 cells but absent on all other thymic i NKT subsets. We further demonstrate that DR3 ligation leads to the in vivo activation of thymic NKT17 cells and provides in vitro costimulatory effects upon α-GalCer-stimulation. Thus, our study reports the identification of a specific surface marker for thymic NKT17 cells that selectively triggers their activation both in vivo and in vitro . These findings provide new insights for deciphering the role and function of IL-17-producing NKT17 cells and for understanding the development and activation mechanisms of i NKT cells in general.

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  1. eLife

    Author Response

    Reviewer #3 (Public Review):

    S Luo and co-workers asked whether NKT17 subset had lineage-specific requirement/s for thymic development beyond what is currently known. Further, they determined what role such a requirement played in activating NKT cells in vivo and in vitro. The strength of the report is the finding that DR3 functions as a selective co-stimulator of NKT17 subset. Experiments appear well-thought out and executed, and the emergent data reasonably carefully interpreted. Some points to consider:

    1. The statement in the abstract and elsewhere that "However, the molecular mechanisms that drive the thymic development and subset-specific activation of NKT17 cells remain mostly unknown" is incorrect. It is better to say, "Much is known yet how this subset develops in the thymus and is activated in the periphery is incompletely understood."

    We thank the reviewer for this suggestion. Accordingly, we have changed the original phrase in the abstract to “How NKT17 cells develop in the thymus and what stimulatory signals would trigger their activation remain incompletely understood. “ (please see line 35-36).

    1. In this regard, if subsets are already formed, why should there be a subset-specific mechanism/s of activation beyond affinity thresholds? The literature suggests that different routes of bacterial inoculation results in the activation of all subsets within a tissue where infection has occurred.

    This is an interesting question. Because all iNKT cells express the canonical invariant TCRα (Vα14-Jα18 in mice), it is reasonable to argue that the same microbial antigen would trigger the activation of all iNKT cells within a given tissue. On the other hand, there are clearly some cytokine receptors and co-stimulatory molecules that are specifically expressed on individual subsets, such as CD122 for NKT1 cells [Lee YJ et al., 2015, Nat. Immunol] and ICOS for NKT2 and NKT17 [Cameron G., 2018, Immunol. Cell Biol] etc., suggesting their role as subset-specific costimulatory molecules. Our identification of DR3 as a thymic NKT17 specific co-stimulatory agent is in support of the perspective that each iNKT subset might require selective co-stimulatory signals for their full activation. We regret that we cannot fully address this question in the current study, but we aim to expand on this question in the near future.

    1. If there is subset specific activation, does this mean that downstream responses from DR3 activation of NKT17 cells prevents the activation of NKT1 & NKT2 subsets? Otherwise, how does one reconcile with the inability of alphaGalCer to activate NKT1 & NKT2 subsets?

    This is an excellent point that we aim to address in our follow-up studies. It is well established that the generation of different iNKT subsets require distinct strengths of TCR signaling, whereby NKT2 cells depend on strong agonistic TCR signals while NKT1 cells are less so. Whether DR3-activated NKT17 cells would prevent or dampen TCR signaling in other iNKT subsets, and whether there is a subset-specific effect of NKT17 cells on NKT1 versus NKT2 cells is unclear to us. However, we will investigate this point as we have now secured DR3-deficient mice for our studies. We are currently in the process of back-crossing DR3-KO mice onto the BALB/c background, which will require several more months; we will then examine this issue in further detail. At this point, we consider assessing this issue as beyond the scope of this manuscript.

    1. The statement "However, the role of CD138 in NKT17 cell biology remains mostly unclear" is incorrect as it was recently reported that CD138 serves are a NKT17 subset-specific marker but the development and function of NKT17 cells do not depend on CD138! So also, results presented herein also supports this view of CD138 about NKT17 cell development and function-nothing new here.

    Here, the reviewer is referring to our statement in line 60, where we are also citing the two publications that have studied the role of CD138 (Syndecan-1) on NKT17 cells in detail. Both studies report that CD138 is specifically expressed on NKT17 cells but that it is not required for their generation. Our statement was intended to highlight this point. To dampen down, we have now modified this sentence to “However, the role of CD138 in NKT17 cell biology is not yet fully understood and remains to be resolved”.

    1. The MS requires proper editing: e.g., "[Please add: Luo S., 2021, JCI Insight];" this incompleteness was found in the introduction.

    We apologize for this oversight. The reference is now properly formatted (line 61), and we have reviewed the revised manuscript multiple times to avoid other mistakes.

    1. Please provide original references to "Because NKT17 cells are the major producers of IL-17 in the thymus and in barrier tissues, such as the lung and skin ..."

    Here, the reviewer is referring to a sentence from the Introduction (line 61-62). The tissue distribution of NKT17 cells has been previously reported, so that we added the original reference [Lee YJ et al., 2015, Immunity] to the text (line 62) as requested.

    1. Whilst "unveiling a new layer of control in NKT17 cell biology" is quite interesting, it is not as surprising as relayed! That NKT cells use second signals to elaborate type I immune responses has been known for at least a couple of decades now.

    We concur that a second signal to control iNKT cell activation has been previously observed and documented. The novelty of our findings, however, lies in the fact that we identified a co-stimulatory signal that is specific to NKT17 cells and that this co-stimulatory signal is conveyed through the cytokine receptor DR3. Thus, we wish to keep this statement in our manuscript, and we hope that the reviewer agrees.

    1. "... we identified the TNF receptor superfamily member 25 (TNFRS25), also known as DR3 (Meylan et al., 2011), being highly expressed on thymic NKT17 cells (Figure 1A and 1B)" while true of BALB/c thymuses, seems less true of C56BL/6 thymuses based on their figure s1. This should be clearly stated in the results. Strain differences in NKT cell content and relative ratios of the subsets are known; hence, it is important to indicate of which strains a particular property/ies is true.

    We appreciate the reviewer’s comments on the strain difference. Prompted by the reviewer’s suggestion, we have expanded our analysis to peripheral organs (LN and lung) of both BALB/c and C57BL/6 mice. These experiments include the co-staining of DR3 and CD138 in thymic iNKT cells in C57BL/6 mice (Figure 1 -figure supplement 3A), and DR3 staining in NKT17 versus non-NKT17 (NKT1 and NKT2 cells) in the thymus, LN and lung of C57BL/6 and BALB/c mice (Figure 1 -figure supplement 3B). Consistent with the data showed by intracellular staining (Figure 1 – figure supplement 1), we found that DR3 is highly expressed on thymic NKT17 cells of both BALB/c and C57BL/6 mice. However, DR3 expression substantially increased on non-NKT17 cells in peripheral tissues, indicating that highly selective DR3 expression on NKT17 cells is rather limited to thymus-resident NKT17 cells. We are now highlighting and explaining these observations in the Results section.

    1. It is indeed surprising that the cytokine profile post alphaGalCer+anti-CD3 stimulation was not assessed.

    In our original submission, we had omitted the cytokine expression part to streamline the narrative and to focus more on the DR3-induced activation part of the thymic iNKT cells. We realize that this was not a wise decision as it raised more questions than explaining our finding. Therefore, we now added data assessing the cytokine production after -GalCer and anti-DR3 stimulation. We found that -GalCer stimulation in the presence of anti-DR3 significantly augmented IL-17 production in thymic NKT17 cells compared to -GalCer stimulation alone (new Figure 3E). Such co-stimulatory effect of DR3 was specific to NKT17 cells and IL-17 production, because we did not find an increase for IL-4 production in non-NKT17 cells (Figure 3 – Figure supplement 5B).

    1. And lastly in a similar vein, a mechanism and the in vivo relevance of this curious co-stimulatory finding remain wanting.

    These are indeed important questions, and the reviewer reiterated these issues below in the “Recommendations for the authors”. Thus, we are providing answers to these issues, e.g. the mechanism and the in vivo relevance, in the section below. Of note, we acquired new mouse models (Nur77-GFP reporter mice and TL1ATg mice) and performed a series of new experiments to address these points, and we hope that these new data have satisfactorily addressed the reviewer’s concern.

  2. eLife

    Evaluation Summary:

    The manuscript by Luo et al. showed that the cytokine receptor DR3 is selectively expressed on thymic NKT17 cells and DR3 ligation leads to the activation of NKT17 cells in the thymus. Overall, The presented experiment are properly executed, controlled and presented. The finding that DR3 acts as a costimulatory molecule for thymic NKT17 cells is interesting. The mechanism and the functional relevance of this finding remain wanting.

    (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 #1 and Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    In this study, the authors showed that the TNF receptor superfamily member Death Receptor-3 (DR3) is preferentially expressed on thymic NKT17 cells in both BALB/c and B6 mice. They further demonstrated that injection of agonistic anti-DR3 can directly activate thymic NKT17 cells in vivo. In addition, DR3 ligation provides costimulatory effects to α-GalCer-stimulated thymic NKT17 cells in vitro. Overall, the experiments are well-performed, and the conclusions are largely supported by the presented data. However, it is not clear whether DR3 ligation affects the function of NKT cells and whether DR3 plays a similar role in human NKT17 cells.

  4. eLife

    Reviewer #2 (Public Review):

    In this manuscript Luo et al. report that amongst invariant Natural Killer T cell subsets that exist, iNKT17 are uniquely expressing the TNF receptor superfamily cytokine receptor DR3 and that DR3 ligation can induce the expression of activation markers (CD69, CD25) on the surface of iNKT17 cells.

    While the finding is certainly new and has not been reported before, data mining from published bulk RNA-expression of iNKT cell subsets or single cell RNA-seq data, had previously established that DR3 transcripts were uniquely found in iNKT17 cells.

    Nonetheless, here, the novelty lies within the demonstration that iNKT17 respond to agonistic treatment with an anti-DR3 antibody.

    The presented experiment are properly executed, controlled and presented.

  5. eLife

    Reviewer #3 (Public Review):

    S Luo and co-workers asked whether NKT17 subset had lineage-specific requirement/s for thymic development beyond what is currently known. Further, they determined what role such a requirement played in activating NKT cells in vivo and in vitro. The strength of the report is the finding that DR3 functions as a selective co-stimulator of NKT17 subset. Experiments appear well-thought out and executed, and the emergent data reasonably carefully interpreted. Some points to consider:
    1. The statement in the abstract and elsewhere that "However, the molecular mechanisms that drive the thymic development and subset-specific activation of NKT17 cells remain mostly unknown" is incorrect. It is better to say, "Much is known yet how this subset develops in the thymus and is activated in the periphery is incompletely understood."
    2. In this regard, if subsets are already formed, why should there be a subset-specific mechanism/s of activation beyond affinity thresholds? The literature suggests that different routes of bacterial inoculation results in the activation of all subsets within a tissue where infection has occurred.
    3. If there is subset specific activation, does this mean that downstream responses from DR3 activation of NKT17 cells prevents the activation of NKT1 & NKT2 subsets? Otherwise, how does one reconcile with the inability of alphaGalCer to activate NKT1 & NKT2 subsets?
    4. The statement "However, the role of CD138 in NKT17 cell biology remains mostly unclear" is incorrect as it was recently reported that CD138 serves are a NKT17 subset-specific marker but the development and function of NKT17 cells do not depend on CD138! So also, results presented herein also supports this view of CD138 about NKT17 cell development and function-nothing new here.
    5. The MS requires proper editing: e.g., "[Please add: Luo S., 2021, JCI Insight];" this incompleteness was found in the introduction.
    6. Please provide original references to "Because NKT17 cells are the major producers of IL-17 in the thymus and in barrier tissues, such as the lung and skin ..."
    7. Whilst "unveiling a new layer of control in NKT17 cell biology" is quite interesting, it is not as surprising as relayed! That NKT cells use second signals to elaborate type I immune responses has been known for at least a couple of decades now.
    8. "... we identified the TNF receptor superfamily member 25 (TNFRS25), also known as DR3 (Meylan et al., 2011), being highly expressed on thymic NKT17 cells (Figure 1A and 1B)" while true of BALB/c thymuses, seems less true of C56BL/6 thymuses based on their figure s1. This should be clearly stated in the results. Strain differences in NKT cell content and relative ratios of the subsets are known; hence, it is important to indicate of which strains a particular property/ies is true.
    9. It is indeed surprising that the cytokine profile post alphaGalCer+anti-CD3 stimulation was not assessed.
    10. And lastly in a similar vein, a mechanism and the in vivo relevance of this curious co-stimulatory finding remain wanting.

  6. Version published to 10.1101/2021.09.27.461992v1 on bioRxiv