The cIAP ubiquitin ligases sustain type 3 γδ T and innate lymphoid cells during aging to allow normal cutaneous and mucosal responses
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Abstract
Environmental and molecular cues early in life are often associated with the permanent shaping of our immune system during adulthood. Although increasing, our knowledge of the signaling pathways that operate in early life and their temporal mode of action is limited. Herein, we demonstrate that the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2), which are E3 ubiquitin ligases and master regulators of the nuclear factor-kappa B (NF-κB) pathway, function during late neonatal and prepubescent life to sustain interleukin(IL)-17-producing gamma delta T cells (γδT17) and group 3 innate lymphoid cells (ILC3). We show that cell-intrinsic deficiency in cIAP1/2 at 3-4 weeks of life leads to downregulation of the transcription factors cMAF and RORγt, and failure to enter cytokine-induced cell cycle. This is followed by progressive loss of γδT17 cells and ILC3 while mice are aging. Mice deficient in cIAP1/2 have severely reduced γδT17 cells and ILC3, present with suboptimal γδT17 responses in the skin, lack small intestinal isolated lymphoid follicles and cannot control intestinal bacterial infection. Mechanistically, these effects appear to be dependent on overt activation of the non-canonical NF-κB pathway. Our data identify the cIAP E3 ubiquitin ligases as critical early life molecular switches for establishing effective type-3 immunity during aging.
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Reply to the reviewers
Point-by-point response
We thank the reviewers for their constructive comments. We have addressed all of them to the best of our knowledge. Our responses are shown below in bold and all changes in the text are highlighted in yellow.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This study of Rizk, Bekiaris and colleagues is well written, carefully edited, and nicely placed into the trending context of the juvenile immune system development.
They suggest that cIAP ubiquitin ligases cIAP1 and cIAP2 sustain type 3 γδ T after 4-5 weeks of age in mice. As a mechanism they show that these ubiquitin ligases are required in a cell-intrinsic …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Point-by-point response
We thank the reviewers for their constructive comments. We have addressed all of them to the best of our knowledge. Our responses are shown below in bold and all changes in the text are highlighted in yellow.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This study of Rizk, Bekiaris and colleagues is well written, carefully edited, and nicely placed into the trending context of the juvenile immune system development.
They suggest that cIAP ubiquitin ligases cIAP1 and cIAP2 sustain type 3 γδ T after 4-5 weeks of age in mice. As a mechanism they show that these ubiquitin ligases are required in a cell-intrinsic manner to maintain cMAF and RORγt levels, and that this depends likely on overt activation of the non-canonical NF-κB pathway.
Extrinsic factors such as microbiota did not seem to play a major role in this context.
**Major comments:**
It is absolutely crucial to directly and stringently control the efficiency of cIAP depletion via RORgt-cre, which may take some time and thus perhaps only reaches relevant (exponential) penetrance at early adulthood?
Fig 4C is nice, however the Birc2 loxP sites may be far less efficient than those in the ROSA26-LSL-RFP system.
__- We thank the reviewer for this comment. In this regard, we sorted day 1 old γδT17 cells from the thymus of Cre+ and Cre- mice and screened for Birc2 mRNA (cIAP1) expression. We additionally compared expression to CD27+ γδ T cells, from the same thymi as RORγt-neg controls. Please see new Fig S5A and text line 208-209. __
However, the pre-puberty timing aspect is surprising, but without this aspect the conclusions would be similarly exciting.
__- The fact that Birc2 is indeed deleted in newborn thymocytes, supports our conclusions that its impact is seen progressively while mice are aging __
**Minor comments:**
- To understand the general impact of cIAP on gdT17 homeostasis, the authors should consider investigating them in additional organs, as these gdT17 are quite tissue-resident and differentially adapt to their environment, where they use specific anti-apoptotic strategies to persist, including expression of Bcl2a1 family proteins.
__- We have investigated lung from adult ΔIAP1/2 and found significantly reduced γδT17 cells, in accordance with our data in the LN, gut and skin. Please see new Fig S1E and text lines 134-136. __
- Fig. 3: Has the presence of gdT17 in the graft been analyzed or enumerated? Experiments shown in Fig 3 AB and FG might collectively suggest that co-transferred gdT17 from the 45.1 BM graft could have reconstituted the regenerated gdT17 compartment in competition with the radioresistant 45.1/2 host gdT17 cells. This would actually not compromise the results, as the cIAP deficient cells did not persist.
__- We are not entirely sure what the reviewer means with this comment. We believe that the data in Fig 3 clearly shows that ΔIAP1/2 cells cannot compete with WT cells. This is also reinforced in Fig S3 where the host is ΔIAP1/2. __
Reviewer #1 (Significance (Required)):
**Significance**
This work is very original and might be of pharmacological interest for approaches targeting cIAP, e.g. in order to enhance anti-viral therapies.
**Referee Cross-commenting**
No further comments.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The authors studied the effect of the inactivation of cIAP1 and 2 on the development and evolution of γδ T cells and in type 3 innate lymphoid cells (ILC3) using RORc-Cre induced inatiction of cIAP1 in combination or not with cIAP2 whole body KO. The authors showed that these two E3 ubiquitin ligases that regulate the NF-κB pathway, are important to maintain a population of IL-17 producers γδ T and ILC3 in adult animals. This lack of maintenance is correlated with a loss of c-MAF and RORγt expression in the two cell types and may be related to a deficiency in entering cell cycle in response to various cytokines. The authors also established that the mechanism is independent of the TNFR1 pathway. The article is well written, clear and most of the conclusions are well supported by the data showed. The results presented are novel and interesting for the field. However, I would suggest some major changes to make the story suitable for publication.
1- The study of 2 different cell types brings some confusion to the story, even if I understand it makes some sense to pool these two parts in the same article. The γδ T cell part is more complete than the ILC3 part, which brings some frustration for the reader, as nothing indicates that the mechanisms leading to the loss of maintenance are similar in the 2 cell types. I would suggest to simply remove the ILC3 part and keep it for another article. If the authors wish to keep it in this article, they must perform a similar set of experiments already done for the γδ T cell part, especially the lineage tracing performed in figure 5 as c-Maf is known to be important in ILC plasticity for ILC3 and ILC1. They would also need to confirm the mechanisms involved in the process leading to ILC3 decrease.
- We thank the reviewer for this comment. We do realize that the ILC3 part of the story may seem incomplete. For this reason, we have taken into consideration the reviewer’s advice and performed lineage tracing in ILC3 cells. In adult ΔIAP1/2 mice that were reporting RFP in RORγt+ cells, we found that within the ILC population, there was 10-fold reduction in RFP+ cells, suggesting that it is unlikely they convert to a non-ILC3 population. Please see new Fig S9C and text lines 297-302. In accordance KLRG1+ ILC2 numbers were not affected (Fig S9C).
__Next, we isolated sLP lymphocytes from 4-week old mice and treated them with cytokines that are known to induce ILC3 proliferation including IL-7, IL-1β and IL-23. We also chose these cytokines to concur with our γδT17 findings. However, we could not induce cell cycle in either WT or ΔIAP1/2 cells. We contacted experts in the field, namely Dr David Withers at the University of Birmingham, who contacted further experts (Dr Matt Hepworth), in order to ask for advice of how to induce gut ILC3 proliferation. We quote David Withers “we have never had any joy making ILC3 proliferate much in vitro”, and Matt Hepworth “have been looking at this and have struggled to make them proliferate in vivo or in vitro”. So, unfortunately, we cannot test ILC3 proliferation in the same way we did for γδT17 cells. __
2- Although the authors nicely excluded the TNFR1 pathway from the mechanisms leading to γδ T cell loss in adult, the overt activation of the cRel pathway is not enough established as far as I am concerned. It would at least require a more thorough quantification of the immunofluorescent staining done. Showing only one cell is not enough. If possible, using another approach to confirm these data would also be needed.
- We have now quantified RelB nuclear translocation over 4 experiments and found a significant increase in ΔIAP1/2 cells. Please see new panel in Fig 5F and text lines 244-250. Furthermore, there was a significant increase in Relb mRNA in ΔIAP1/2 newborn thymic γδT17 cells, which is consistent with activation of the non-canonical NF-kB pathway. Please see new Fig S6C and text lines 244-246.
3- The expression level/quantity of protein of cIAP1/2 in γδ T cells from WT animal at the various stages of development has not been analyzed. Does it remain constant? Does it vary throughout development of γδ T cells? This information is important to further enforce and understand the role of these protein in the development of γδ T cells.
__- Unfortunately, we cannot quantify cIAP1/2 protein levels in these cells for technical reasons. There is no Ab for flow and only a cIAP1 Ab for western blots, which is of course impossible when dealing with such low cell numbers.____ However, we have contacted Dr Dominic Grün who had done a single-cell RNA-seq profiling of γδ T cells throughout different developmental stages, and asked to analyze expression levels of Birc2 and Birc3. We found that both Birc2 and Birc3 were expressed across all subsets of fetal and adult thymic γδ T cells with no specific enrichment and no apparent up- or down-regulation between the two time points. Please see attached Figure 1. __
Attached Figure 1: expression patterns of Birc2 and *Birc3 *at a single cell level in the different populations of fetal and adult thymic γδ T cells.
4- In Figure 4D-E, the authors showed that in vitro, γδ T cells fail to progress through cell cycle in response to IL-7 or IL1b+ IL-23. Is a similar block detectable directly ex-vivo? Furthermore, it appears that Imiquimod treatment restore at least partially the deficiency in γδ T cells in the double KO mice. It would mean other cytokines or TCR triggering is rescuing this phenotype. Could the author test in vitro other stimuli and test whether γδ T cells are reactive to some stimuli but not others? It would bring some lights on the signaling regulated by cIAP1/2.
- There is little if any detectable active cell cycling of these cells directly ex vivo, as shown by near absence of Ki67+7AAD+ cells (see below). We can still pick up small differences in Ki67+ cells but this is not sufficient to conclude whether there is more or less cell division. Please see attached figure 2.
Attached Figure 2: ex-vivo cell cycle analysis of γδT17 cells form 4 week old- ΔIAP2 or ΔIAP1/2 mice.
__- We have now tested how 4-week old γδT17 cells from ____Δ____IAP1/2 mice respond to IL-2 and TCR stimulation. We found that similar to IL-7, IL-1b and IL-23, cells lacking IAPs proliferate less under both conditions. See new Fig S5C and lines 227-228. __
**Minor point:**
although the authors cite a reference in the result section, could they show a dot plot confirming that the CD44hi CCR6+ or CCR6+ are the only population producing IL-17 among, γδ T cells?
- We now show this in Fig S1D and lines 129-130
Reviewer #2 (Significance (Required)):
This article describes a new role for the cIAP1 and 2 in the maintenance of γδ T cells and ILC3s. In line with their previous work (Rizk, 2019), they show that this effect is correlated with a loss in c-MAF expression, which is a major transcription factor for these 2 cell types. These discoveries are of interest for specialists in the field, including myself. I am an expert in T cells and ILCs, with an interest in c-MAF function in these cell types.
**Referee Cross-commenting**
No further comments.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The authors studied the effect of the inactivation of cIAP1 and 2 on the development and evolution of T cells and in type 3 innate lymphoid cells (ILC3) using RORc-Cre induced inatiction of cIAP1 in combination or not with cIAP2 whole body KO. The authors showed that these two E3 ubiquitin ligases that regulate the NF-B pathway, are important to maintain a population of IL-17 producers T and ILC3 in adult animals. This lack of maintenance is correlated with a loss of c-MAF and RORγt expression in the two cell types and may be related to a deficiency in entering cell cycle in response to various cytokines. The authors also …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The authors studied the effect of the inactivation of cIAP1 and 2 on the development and evolution of T cells and in type 3 innate lymphoid cells (ILC3) using RORc-Cre induced inatiction of cIAP1 in combination or not with cIAP2 whole body KO. The authors showed that these two E3 ubiquitin ligases that regulate the NF-B pathway, are important to maintain a population of IL-17 producers T and ILC3 in adult animals. This lack of maintenance is correlated with a loss of c-MAF and RORγt expression in the two cell types and may be related to a deficiency in entering cell cycle in response to various cytokines. The authors also established that the mechanism is independent of the TNFR1 pathway. The article is well written, clear and most of the conclusions are well supported by the data showed. The results presented are novel and interesting for the field. However, I would suggest some major changes to make the story suitable for publication.
1- The study of 2 different cell types brings some confusion to the story, even if I understand it makes some sense to pool these two parts in the same article. The T cell part is more complete than the ILC3 part, which brings some frustration for the reader, as nothing indicates that the mechanisms leading to the loss of maintenance are similar in the 2 cell types. I would suggest to simply remove the ILC3 part and keep it for another article. If the authors wish to keep it in this article, they must perform a similar set of experiments already done for the T cell part, especially the lineage tracing performed in figure 5 as c-Maf is known to be important in ILC plasticity for ILC3 and ILC1. They would also need to confirm the mechanisms involved in the process leading to ILC3 decrease.
2- Although the authors nicely excluded the TNFR1 pathway from the mechanisms leading to T cell loss in adult, the overt activation of the cRel pathway is not enough established as far as I am concerned. It would at least require a more thorough quantification of the immunofluorescent staining done. Showing only one cell is not enough. If possible, using another approach to confirm these data would also be needed.
3- The expression level/quantity of protein of cIAP1/2 in T cells from WT animal at the various stages of development has not been analyzed. Does it remain constant? Does it vary throughout development of T cells? This information is important to further enforce and understand the role of these protein in the development of T cells.
4- In Figure 4D-E, the authors showed that in vitro, T cells fail to progress through cell cycle in response to IL-7 or IL1b+ IL-23. Is a similar block detectable directly ex-vivo? Furthermore, it appears that Imiquimod treatment restore at least partially the deficiency in T cells in the double KO mice. It would mean other cytokines or TCR triggering is rescuing this phenotype. Could the author test in vitro other stimuli and test whether T cells are reactive to some stimuli but not others? It would bring some lights on the signaling regulated by cIAP1/2.
Minor point:
although the authors cite a reference in the result section, could they show a dot plot confirming that the CD44hi CCR6+ or CCR6+ are the only population producing IL-17 among , T cells?
Significance
This article describes a new role for the cIAP1 and 2 in the maintenance of T cells and ILC3s. In line with their previous work (Rizk, 2019), they show that this effect is correlated with a loss in c-MAF expression, which is a major transcription factor for these 2 cell types. These discoveries are of interest for specialists in the field, including myself. I am an expert in T cells and ILCs, with an interest in c-MAF function in these cell types.
Referee Cross-commenting
No further comments.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This study of Rizk, Bekiaris and colleagues is well written, carefully edited, and nicely placed into the trending context of the juvenile immune system development.
They suggest that cIAP ubiquitin ligases cIAP1 and cIAP2 sustain type 3 γδ T after 4-5 weeks of age in mice. As a mechanism they show that these ubiquitin ligases are required in a cell-intrinsic manner to maintain cMAF and RORγt levels, and that this depends likely on overt activation of the non-canonical NF-κB pathway. Extrinsic factors such as microbiota did not seem to play a major role in this context.
Major comments:
It is absolutely crucial to directly and …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This study of Rizk, Bekiaris and colleagues is well written, carefully edited, and nicely placed into the trending context of the juvenile immune system development.
They suggest that cIAP ubiquitin ligases cIAP1 and cIAP2 sustain type 3 γδ T after 4-5 weeks of age in mice. As a mechanism they show that these ubiquitin ligases are required in a cell-intrinsic manner to maintain cMAF and RORγt levels, and that this depends likely on overt activation of the non-canonical NF-κB pathway. Extrinsic factors such as microbiota did not seem to play a major role in this context.
Major comments:
It is absolutely crucial to directly and stringently control the efficiency of cIAP depletion via RORgt-cre, which may take some time and thus perhaps only reaches relevant (exponential) penetrance at early adulthood? Fig 4C is nice, however the Birc2 loxP sites may be far less efficient than those in the ROSA26-LSL-RFP system.
However, the pre-puberty timing aspect is surprising, but without this aspect the conclusions would be similarly exciting.
Minor comments:
- To understand the general impact of cIAP on gdT17 homeostasis, the authors should consider investigating them in additional organs, as these gdT17 are quite tissue-resident and differentially adapt to their environment, where they use specific anti-apoptotic strategies to persist, including expression of Bcl2a1 family proteins.
- Fig. 3: Has the presence of gdT17 in the graft been analyzed or enumerated? Experiments shown in Fig 3 AB and FG might collectively suggest that co-transferred gdT17 from the 45.1 BM graft could have reconstituted the regenerated gdT17 compartment in competition with the radioresistant 45.1/2 host gdT17 cells. This would actually not compromise the results, as the cIAP deficient cells did not persist.
Significance
Significance
This work is very original and might be of pharmacological interest for approaches targeting cIAP, e.g. in order to enhance anti-viral therapies.
Referee Cross-commenting
No further comments.
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