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  1. Author Response:

    Reviewer #1 (Public Review):

    This manuscript has great potential. The study is well designed, performed, and written, with good statistical analyses. On the other hand, it does not have a sufficient experimental basis. The authors investigated whole body immunoglobulin diversity in killifish and found that it decreases with age. This decrease is mostly driven by larger clones, in other words, by the expansion of B cell clones. They further analyzed immunoglobulin diversity in the intestine and found that its decrease is much more pronounced than in the whole body. It was also observed that the transfer of the young gut flora to old fish does not rejuvenate the B cell repertoire. The major novelty of this work is the model organism, killifish. Also, while this study is solid, it is descriptive, without many mechanistic insights.

    We thank the reviewer for their frank assessment of our manuscript, as well as for their helpful suggestions of possible ways to dig deeper into the phenomenon of killifish repertoire ageing. We agree with the assessment that this study is primarily descriptive in nature, and that experimental interventions – including infection challenge studies – would help establish causal mechanisms. Nevertheless, we have provided new data supporting an association between loss of repertoire diversity (see our response below) which we believe supports the biological relevance of our findings.

    While our initial submission demonstrates that the diversity of the killifish repertoire declines with age, it is true that this does not necessarily imply that this decline is linked to changes in immune functionality. To provide functional insights into the transcriptomic signature associated with different antibody diversity orders, we now include an analysis linking repertoire diversity data in our intestinal cohort to pre-existing intestinal RNA-seq data from the same individuals (Figure 6). The combination of these two data sets allows us to analyse changes in gene expression with respect to intestinal antibody diversity, controlling for age. We find that a number of immune-activity GO terms – including “B cell receptor signaling pathway”, “B cell proliferation”, and “lymphocyte activation” are significantly positively enriched with respect to repertoire diversity across multiple diversity orders. A decline in intestinal antibody diversity – as seen in ageing – is thus associated with a decline in B-cell immune activity in killifish. We acknowledge that confident demonstration of a causal link between repertoire diversity and immune state will require experimental challenge of host immunity, for example through infection experiments – something we will address in the future and is beyond the scope of this work. However, we believe these new data are sufficient to demonstrate a significant association between the two, supporting the biological relevance of the age-associated decline in diversity we observe.

    Some of the following experiments, or other experiments, may help explore mechanisms and make the study more compelling: 1) whole genome sequencing of lymphoid tissues and brain as a control, from the same old fish to determine whether there are clonal somatic mutations. If confirmed, it may be an important finding, as it would mean that clonal expansions emerge as fast as the killifish lifespan, and it would be a great model to study mechanisms of mutation accumulation and clonal selection with age. This WGS data may be further used to reconstruct immunoglobulin repertoires to understand if the whole-body decrease is driven solely by intestine B cells, or it initiates in lymphoid tissues.

    We agree that further investigation of primary repertoire development in killifish lymphoid organs would be a valuable direction for future work, and would help disentangle whole-body from intestinespecific repertoire changes. However, we believe our current analysis is sufficient to demonstrate the presence of clonal somatic mutations in the whole-body repertoire. The pRESTO/Change-O pipeline used in our analysis can distinguish heavy-chain sequences arising from different naive ancestors, and the presence of large clones in the killifish repertoire (see e.g. Supplemental Figure 5A) necessitates rapid clonal expansion. Ongoing work in our group is indeed directed at studying somatic DNA sequence variation across tissues during aging in killifish, including alternative experimental approaches to investigating killifish repertoire aging. We have now added a sentence about these further research directions to the manuscript discussion. However, we feel these further experiments may be beyond the specific scope Bradshaw et al. Point to point rebuttal of the present work, which is focused on high-level changes in killifish antibody repertoire composition with age.

    1. RNA sequencing of intestine samples or spleen from young versus old killifish to obtain insights into possible molecular mechanisms clonal expansion and diversity loss. Spleen RNA sequencing may be used to reconstruct the immunoglobulin repertoire. The authors used 750 ng of total RNA in the current study, so there should be enough material for RNA sequencing. As an alternative, single cell RNA sequencing may be performed.

    We certainly agree that investigation of repertoire aging in a wider array of immune organs, including spleen, would be highly valuable, and that killifish is a promising model organism in which to carry out these investigations. We have now included analysis of RNA-sequencing data from the killifish gut, which as discussed above supports an association between loss of repertoire diversity and immune function in that organ (see response to A.1). We hope for future work to more comprehensively explore the landscape of organ-specific repertoire ageing in the turquoise killifish; however, we feel that this would be beyond the scope of the present study.

    Reviewer #2 (Public Review):

    This study introduces the killifish as a short-lived vertebrate model for immune aging and immunosenescence and characterizes the changes in the immune-repertoire during aging. The authors convincingly show a decrease in diversity of the large expanded B-cell clones that is greater than small clones and a more pronounced change in the intestinal antibody repertoire with age. A limitation of the current study is its descriptive nature and lack of strong evidence that these animals truly experience functional immunosenescence. The impact of this work could be strengthened by functional data showing a decline in adaptive immunity that goes along with the loss of diversity in the antibody repertoire or citation and discussion of prior literature supporting this relationship. As it is, it is difficult to know the extent to which the observed changes are strongly correlated with changes in immune function, and the manuscript currently somewhat overstates the importance of the observations. It should be explicitly noted that further research is needed to determine whether the changes in immune-repertoire actually reflect immune senescence or simply changes with little or no consequence.

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  2. Evaluation Summary:

    This study introduces the killifish as a potential model for immune aging and immunosenescence and characterizes the changes in age-associated immune-repertoire. The authors convincingly show a decrease in diversity of the large expanded B-cell clones that is greater than small clones and a more pronounced change in the intestinal antibody repertoire with age. These results strongly suggest that killifish undergo age-related immunosenescence. Adding functional measures of the immune system would strengthen this conclusion.

    (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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  3. Reviewer #1 (Public Review):

    This manuscript has great potential. The study is well designed, performed, and written, with good statistical analyses. On the other hand, it does not have a sufficient experimental basis. The authors investigated whole body immunoglobulin diversity in killifish and found that it decreases with age. This decrease is mostly driven by larger clones, in other words, by the expansion of B cell clones. They further analyzed immunoglobulin diversity in the intestine and found that its decrease is much more pronounced than in the whole body. It was also observed that the transfer of the young gut flora to old fish does not rejuvenate the B cell repertoire. The major novelty of this work is the model organism, killifish. Also, while this study is solid, it is descriptive, without many mechanistic insights.

    Some of the following experiments, or other experiments, may help explore mechanisms and make the study more compelling: 1) whole genome sequencing of lymphoid tissues and brain as a control, from the same old fish to determine whether there are clonal somatic mutations. If confirmed, it may be an important finding, as it would mean that clonal expansions emerge as fast as the killifish lifespan, and it would be a great model to study mechanisms of mutation accumulation and clonal selection with age. This WGS data may be further used to reconstruct immunoglobulin repertoires to understand if the whole-body decrease is driven solely by intestine B cells, or it initiates in lymphoid tissues. 2) RNA sequencing of intestine samples or spleen from young versus old killifish to obtain insights into possible molecular mechanisms clonal expansion and diversity loss. Spleen RNA sequencing may be used to reconstruct the immunoglobulin repertoire. The authors used 750 ng of total RNA in the current study, so there should be enough material for RNA sequencing. As an alternative, single cell RNA sequencing may be performed.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    This study introduces the killifish as a short-lived vertebrate model for immune aging and immunosenescence and characterizes the changes in the immune-repertoire during aging. The authors convincingly show a decrease in diversity of the large expanded B-cell clones that is greater than small clones and a more pronounced change in the intestinal antibody repertoire with age. A limitation of the current study is its descriptive nature and lack of strong evidence that these animals truly experience functional immunosenescence. The impact of this work could be strengthened by functional data showing a decline in adaptive immunity that goes along with the loss of diversity in the antibody repertoire or citation and discussion of prior literature supporting this relationship. As it is, it is difficult to know the extent to which the observed changes are strongly correlated with changes in immune function, and the manuscript currently somewhat overstates the importance of the observations. It should be explicitly noted that further research is needed to determine whether the changes in immune-repertoire actually reflect immune senescence or simply changes with little or no consequence.

    Read the original source
    Was this evaluation helpful?