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NFATc1 deficiency in T cells protects mice from experimental autoimmune encephalomyelitis

NFATc1 is a member of the nuclear factor of activated T cells (NFAT) family of transcription factors. NFAT is activated upon T‐cell receptor activation followed by intracytoplasmatic calcium influx where calmodulin, a calcium sensor protein, activates the phosphatase calcineurin that dephosphorylates NFAT proteins and results in NFAT nuclear import. Here, we show the analysis of conditional NFATc1‐deficient mice bearing a deletion of NFATc1 in CD4+ and CD8+ T cells. NFATc1‐deficient CD4+ T cells polarized under Th17 conditions express reduced levels of the Th17‐associated transcription factor RORγT (where ROR is RAR‐related orphan receptor) as well as the Th17‐associated cytokines IL‐17A, IL‐17F, IL‐21, and IL‐10. In the murine model of experimental EAE, we found a strong reduction of the disease outcome in conditional NFATc1‐deficient mice, as compared with control littermates. This was accompanied by a diminished inflammation in the brain and spinal cord and reduced IL‐17A and IFN‐γ expression by antigen‐specific spleen, spinal cord, and brain cells. Altogether, these results reveal an important role of NFATc1 in inducing Th17‐cell responses and IFN‐γ, both being relevant for the EAE development.

Il21, Il23r and Il10 mRNAs, and secrete less IL-17A, IL-17F and IL-10 cytokines (Figs. 1 and 2). Since for Nfatc1-/-Th17 cells they also detected a marked reduction in Rorc mRNA and in number of RORgT cellsbut not for further transcription factors testedone may conclude that NFATc1 supports the development of Th17 cells through RORgT. The opposite seems to be the case for Eomes that was found to be enhanced in Nfatc1-/-Th17 cells. Finally, the authors studied the RNA levels and lymphokine secretion as well as the proliferation of CD4+ T cells from brain and spinal cord (but mostly from spleen) of mice upon EAE induction.
They observed a marked reduction in IL-17A and IFN-g as well as IL-2 and TNF-a levels. Unfortunately, they did not investigate the levels of transcription factors, which they described for Th17 cells, i.e. for RORgT, Eomes and several others.
Taken together, the major findingthe role of NFATc1 in controlling EAE through affecting Th17 lymphokine expression and, probably, Th17 function in generalmight deserve publication in a renowned journal like EJI. However, the experimental results shown here are incomplete, several controls are missing and the text has been written in a quite sloppy style by a person who is not very familiar with the latest (and former!) findings about NFAT transcription factors.
Critical points that have to be corrected, changed or completed: 1. The introductory statement 'The role of NFATc1 in Th17 differentiation processes was not fully investigated so far' appears to be right. However, the efforts by numerous other authors should be mentioned here. They were reviewed by  and (Gomez-Rodriguez et al., 2009) and, in part, by (Vaeth et al., 2012) as a later report.
2. Immunologist should be aware that due to the activity of Cd4-cre in double-positive thymocytes floxed genes become inactivated in both CD4+ and CD8+ T cells (see above and below). In this context, because -cre mice should be replaced by NFATc1ΔT/ΔT or a similar abbreviation (to indicate [whole!] T cell specificity).
3. For scientists doing research on NFAT transcription factors, it should really be common knowledge that calcineurin -and not "kinases" (whose action is exactly the opposite)as outlined in the Introduction (page 3) is the major player in NFATc activation, which becomes activated upon immune receptor activation and de-phosphorylates the NFATc proteins (this is textbook knowledge!).

The authors should cite and discuss that a similar role in MOG-induced EAE has been published in 2011
for NFATc1 (Bhattacharyya et al., 2011), when it was inactivated in splenic B cells. Moreover, the authors of the B cell paper had connected the protection with an enhanced IL-10 production by NFATc1-/-B cells. The presumable different regulation of IL-10 in B and T cells should be discussed. 5. In my view, the entire "Introduction" chapter has to be carefully re-written since the statements about (i) "the main forms expressed in T cells are…" (ii) the "involvement of NFATc1 in Th1/Th2 differentiation" reflect only half of the truth and have been investigatedor correctedin recent publications. 6. A puzzle to the reviewer is the following statement: "Other cytokines known to be highly induced under Th17 cell conditions are ... IL-10" (page 5, results). The only given citation #40 (see page 10) does not mention Th17 cells. An original paper describing the conditions for IL-10 induction in Th17 cells would be (Xu et al., 2009). However, it should be addressed that the field tries to distinguish between pathogenic and non-pathogenic Th17 cells (see (Kurebayashi et al., 2013)). Therefore, firstly it has to be rephrased "highly induced under non-pathogenic Th17 cell conditions, namely in the presence of TGFβ" and secondly the distinction between both Th17 subtypes has to be introduced and discussed. This is very important, because the in vitro generated Th17 cells in the presence of TGFβ might be distinct from the in vivo ones mediating EAE. 7. For the convenience of the future readership, please, add some citations, for example to underpin statements like "EAE is also characterized by an enhanced IFN-γ expression" and "cytokines IL-6 and IL-2, known to support EAE development" (page 7). 10. The title "Reduced proliferation and Th17 cytokine secretion in NFATc1-deficient CD4+ T cells" for describing Fig. 5 in the results is misleading, since it deals with Th1 cells and IFN-y expression/secretion. 11. When regulatory T cells are mentioned, please, make it clear that these are iTreg cells. Here, you should discuss the publication, which demonstrated at least a mild effect on in vitro iTreg differentiation upon NFATc1 deficiency (Vaeth et al., 2012).
Missing experiments that should be performed: 1. NFATc2 expression -compared to NFATc1 -in Th17 and Tc17 cells as well as Th1 and Tc1 cells should be evaluated.
2. Please determine and include statistical significance in all supplementary figures, for example to underline that Maf and Batf are really unchanged as stated.
3. In addition to the shown cumulative RNA and ELISA data, cells from the CNS of EAE animals should be analyzed by intracellular FACS, especially via double-stainings for IL-17A and IFNγ, gated for CD4. This will determine if the observed loss of IFNγ occurs in the pathogenic Th17/1 cells or not. This has to be compared to similar staining of the in vitro generated Th17 and Th1 cells. To distinguish between non-and pathogenic Th17, in vitro cultures should additionally be performed without TGFβ, but in the presence of Il-1β and IL-23.
This will address the different effects of NFATc1 deficiency on IFNγ in vitro and in vivo. 4. Furthermore, as the main point of the authors is the role of NFATc1 in Th17 differentiation, the authors should include the cytokines IL-10 and IL-21 (described in vitro) in FACS analyses as well as transcription factor evaluations for Rorc, Tbx21, Maf and Eomes from ex vivo EAE-cells.
5. Frequencies of CD4+ T cells, CD8+ T cells and B cells as well as the number of antigen-specific CD4+ T cells by tetramer or CD40L  in CNS should be determined. Maybe cells do not reach the CNS, because NFATc1 influences homing receptor expression and not merely due to hampered proliferation.
6. There is evidence that similar to Th17 cells Tc17 cells are regulated by Ca2+ and NFAT. Furthermore, they are important for the Th17-mediated pathogenicity of EAE (Huber et al., 2013). Due to Cd4-cre, also CD8+ T cells have ablated NFATc1. Therefore this population has to be analyzed in parallel. Please, differentiate NFATc1-sufficient and -deficient CD8+ T cells in vitro towards Tc17 and Tc1 and determine IL-17 and IFNγ expression. In this line, when isolating cells from CNS and staining for CD4, IL-17 and IFNγ, please include CD8, IL-17 and IFNγ intracellular FACS.

Reviewer: 2
Comments to the Author In this paper the authors investigate the role of NFATc1 in Th17 differentiation and demonstrate its involvement in EAE disease since NFATc1-deficient mice show a strong reduction of symptoms. Data related to Th17 phenotype, including both cytokines and transcription factors, are clear and well discussed.
The most weakness of the paper regards Th1 phenotype and IFNg production; in fact IFNg reduction observed in supernatants of MOG-restimulated splenic cells (both total and CD4+ T cells) and also in T cells derived from brain and spinal cord, is in contrast with data shown at the beginning of the paper, indicated Moreover the paper needs to be improved answering the following questions.
Major point: 1) Could be the reduced proliferation and cytokines production of MOG-specific splenic cells, related to the low amount of IL-2 produced by the same cells? Will be this proliferation and cytokine restored if rcIL-2 will be added to MOG-specific proliferation assay? What is the effect in terms of proliferation rate and cytokines production if NFATc1-deficient splenic cells will be TCR policlonally stimulated instead of antigen specific stimulation?
2) What about cytokines levels in the supernatants of proliferation of MOG-specific lymph node cells performed on day 17 after EAE induction? Both IL-17 and IFN-g are reduced also in these conditions? This work presents a novel role for transcription factor NFATc1 in Th17 cells, using a T-cell conditional KO for NFATc1. NFATc1-deficient CD4 T cells polarized under Th717 conditions have less Th17 cytokines and less RORgt expression. An in vivo model was performed to demonstrate further this function. An EAE experiment was performed and analyzed quite completely, also analyzing data from brain and spinal cord samples. Their results also point out that IFNg expression is also altered by NFATc1 in this mouse model. Altogether, they nicely conclude that NFATc1 induces Th17 differentiation. Comments: -In the Abstract, Introduction and Results it is mentioned that NFATc1 flox/flox CD4-Cre+ mice show NFATc1-deficiency in CD4-expressing cells. CD4 is expressed at the DP stage, which means that the Cre recombinase will be expressed FROM the DP stage, deleting NFATc1 since some point after that and in both CD4 and CD8 single-positive and mature T cells. Not only CD4 cells will be deficient in NFATc1, but also CD8 cells. At least deletion in both subsets has been seen in most gene-deletions by CD4-Cre.
If these authors really only see deletion of NFATc1 in CD4 T cells, they should show a WB detecting NFATc1 in CD8 cells. If not, the writing should be changed to reflect deletion in both CD4 and CD8 T cells.
-Results shown in Suppl Fig. S2C demonstrate that, in vitro, there is a reduction in IFNg protein expression but not in RNA. They state that they "did not find any significant differences" but they should comment on the protein result, that does show a decrease (page 6, line 27).
-In the Discussion authors comment on the potential binding sites they see on the promoters of the cytokine genes but do not show any ChIP data to indicate their importance. However, they do not make any comment on RORgt, which is the master regulator, and which also shows to be affected by deletion of NFATc1. This should be rectified. The key finding presented in this manuscript is the role of transcription factor NFATc1 in Experimental Autoimmune Encephalomyelitis (EAE), the most commonly used mouse model for Multiple Sclerosis of man. By using Nfatc1fl/fl mice which were crossed with Cd4-cre mice for the ablation of NFATc1 in T cells (in both CD4+ and CD8+ T cells, and not only in CD4+ cells as mentioned several times in the text including the abstract) the authors observed a strong decrease in the course of MOG-peptide induced EAE (Fig. 3C). This appears to be due to a defect in the function of Th17 cells that are assumed to play a major role in EAE induction and maintenance. Thus, upon differentiation of naïve splenic B cells in vitro to Th17 cells (and Th0 control cells) for 4 days the authors show that Th17 cells deficient for NFATc1 synthesize less Il7a, Il17f, Il21, Il23r and Il10 mRNAs, and secrete less IL-17A, IL-17F and IL-10 cytokines ( Figs. 1 and 2). Since for Nfatc1-/-Th17 cells they also detected a marked reduction in Rorc mRNA and in number of RORgT cellsbut not for further transcription factors testedone may conclude that NFATc1 supports the development of Th17 cells through RORgT. The opposite seems to be the case for Eomes that was found to be enhanced in Nfatc1-/-Th17 cells. Finally, the authors studied the RNA levels and lymphokine secretion as well as the proliferation of CD4+ T cells from brain and spinal cord (but mostly from spleen) of mice upon EAE induction.
They observed a marked reduction in IL-17A and IFN-g as well as IL-2 and TNF-a levels. Unfortunately, they did not investigate the levels of transcription factors, which they described for Th17 cells, i.e. for RORgT, Eomes and several others.
Taken together, the major findingthe role of NFATc1 in controlling EAE through affecting Th17 lymphokine expression and, probably, Th17 function in generalmight deserve publication in a renowned journal like EJI. However, the experimental results shown here are incomplete, several controls are missing and the text has been written in a quite sloppy style by a person who is not very familiar with the latest (and former!) findings about NFAT transcription factors.

Answer:
The authors thank the reviewer for his/her contribution to this work. Critical points have been corrected, changed or completed as described below: 1. The introductory statement 'The role of NFATc1 in Th17 differentiation processes was not fully investigated so far' appears to be right. However, the efforts by numerous other authors should be mentioned here. They were reviewed by  and (Gomez-Rodriguez et al., 2009) and, in part, by (Vaeth et al., 2012) as a later report.
Answer: The Reviewer is right. We have now integrated the mentioned papers. The paper from Gomez-Rodriguez was already cited in the originally submitted version of the manuscript.
2. Immunologist should be aware that due to the activity of Cd4-cre in double-positive thymocytes floxed genes become inactivated in both CD4+ and CD8+ T cells (see above and below). In this context, because it implies an inactivation of NFATc1 in all cells, the abbreviation NFATc1Δ Δ for the Nfatc1fl/fl x Cd4-cre mice should be replaced by NFATc1ΔT/ΔT or a similar abbreviation (to indicate [whole!] T cell specificity).
Answer: We are aware that the inactivation affects both CD4+ and CD8+ T cells. We have now integrated clearly this point in the manuscript. In addition, we used, in the previous submission, the label of the mice given in the original paper where the mice were originally described. We changed now the abbreviation NFATc1Δ/Δ to NFATc1Δ T/ΔT in all figures, as suggested.
3. For scientists doing research on NFAT transcription factors, it should really be common knowledge that calcineurin -and not "kinases" (whose action is exactly the opposite)as outlined in the Introduction (page 3) is the major player in NFATc activation, which becomes activated upon immune receptor activation and de-phosphorylates the NFATc proteins (this is textbook knowledge!). √ Answer: Following the reviewer indications we have now integrated Calcineurin as the phosphatase dephosphorylating NFATc1 in the introduction.
4. The authors should cite and discuss that a similar role in MOG-induced EAE has been published in 2011 for NFATc1 (Bhattacharyya et al., 2011), when it was inactivated in splenic B cells. Moreover, the authors of the B cell paper had connected the protection with an enhanced IL-10 production by NFATc1-/-B cells. The presumable different regulation of IL-10 in B and T cells should be discussed. √ Answer: We have now integrated the paper in the discussion and also discussed the presumable different regulation of IL-10 in B-and T cells.
5. In my view, the entire "Introduction" chapter has to be carefully re-written since the statements about (i) "the main forms expressed in T cells are…" (ii) the "involvement of NFATc1 in Th1/Th2 differentiation" reflect only half of the truth and have been investigatedor correctedin recent publications.
Answer: Following the reviewer indications' we have now integrated the recent publications in the introduction. 6. A puzzle to the reviewer is the following statement: "Other cytokines known to be highly induced under Th17 cell conditions are ... IL-10" (page 5, results). The only given citation #40 (see page 10) does not mention Th17 cells. An original paper describing the conditions for IL-10 induction in Th17 cells would be (Xu et al., 2009). However, it should be addressed that the field tries to distinguish between pathogenic and non-pathogenic Th17 cells (see (Kurebayashi et al., 2013)). Therefore, firstly it has to be rephrased "highly induced under non-pathogenic Th17 cell conditions, namely in the presence of TGFβ" and secondly the distinction between both Th17 subtypes has to be introduced and discussed. This is very important, because the in vitro generated Th17 cells in the presence of TGFβ might be distinct from the in vivo ones mediating EAE. √ Answer: We have now integrated the original paper from Xu et. al. 2009 to advise to IL10 expression by Th17 cells. We have also mentioned and discussed the differences between pathogenic and non-pathogenic Th17 cells in the introduction and discussion. 7. For the convenience of the future readership, please, add some citations, for example to underpin statements like "EAE is also characterized by an enhanced IFN-γ expression" and "cytokines IL-6 and IL-2, known to support EAE development" (page 7).
Answer: We have now integrated more publications underlining the role of different cytokines in EAE development in the introduction and the discussion part of the paper . 3a. In addition to the shown cumulative RNA and ELISA data, cells from the CNS of EAE animals should be analyzed by intracellular FACS, especially via double-stainings for IL-17A and IFNγ, gated for CD4. This will determine if the observed loss of IFNγ occurs in the pathogenic Th17/1 cells or not.
Answer: We analyzed the cells from the CNS at day also by trend a reduced IL17A expression in CD4+ T cells ( Figure 7B, D). The number of IFNg+IL17A+ CD4+ T cells in the CNS tend to be enhanced in the absence of NFATc1, but we were not able to detect a -positive T cells in the CNS of these mice via FACS analysis. This has to be further investigated.
3b…..This has to be compared to similar staining of the in vitro generated Th17 and Th1 cells.
Answer: With in vitro generated Th17 and Th1 cells we also did FACS analysis and stained for IL17A and analysis of Th1 or Th17 cell cultures.
3c. To distinguish between non-and pathogenic Th17, in vitro cultures should additionally be performed without TGFβ, but in the presence of Il-1β and IL-23. This will address the different effects of NFATc1 deficiency on IFNγ in vitro and in vivo.
afterwards analyzed the expression of Th17 genes. We found a reduced expression of Rorc mRNA and -deficient pathogenic Th17 cells as compared to control Th17 cells. The expression of Il10, Il17f and Il23r mRNA and IL17F protein was not altered. In contrast, the expression of Il17a was enhanced in the absence of NFATc1 in cells cultured under pathogenic Th17 cell -pathogenic polarized NFATc1-deficient Th17 T cells as compared to control cells (S1-H).
4. Furthermore, as the main point of the authors is the role of NFATc1 in Th17 differentiation, the authors should include the cytokines IL-10 and IL-21 (described in vitro) in FACS analyses as well as transcription factor evaluations for Rorc, Tbx21, Maf and Eomes from ex vivo EAE-cells.
Answer: We have analyzed the expression pattern of ex vivo EAE cells: the expression of Rorc was reduced by trend in NFATc1-deficient CNS cells, whereas Tbx21and Il21 expression levels were significantly diminished ( Figure 6G, H, J). In the expression pattern of Maf and Eomes we were not able to detect differences (S5-F and data not shown). According to the limitation of CNS cells we analyzed the expression of IL10 by lymph node cells. We found a reduced number of CD4+IL10+ T cells in the CNS of NFATc1-deficient mice as compared to control mice. The number of IL10-expressing CD4+ T cells was also reduced by trend ( Figure 9D, E).  in CNS should be determined. Maybe cells do not reach the CNS, because NFATc1 influences homing receptor expression and not merely due to hampered proliferation.

Frequencies of CD4+ T cells, CD8+ T cells and B cells as well as the number of antigen-specific CD4+ T cells by tetramer or CD40L
Answer: Analysis of the frequencies of the above mentioned cell subsets in the CNS showed that in conditional NFATc1-deficient mice there are lower numbers of CD4+ ( Figure 7E) and CD8+ T cells in the CNS, whereas the number of B220+ B lymphocytes was enhanced in the CNS of these mice (S5-B,C). We were not able to detect CD4+CD40L/CD154 double-positive cells in the CNS via FACS analysis. Because of this we did not further discuss that above mentioned point. It is possible that, in addition to the proliferation defect in these cells also the expression of homing receptors might be different leading to the reduced capability for CD4+ T cells to reach the CNS. This has to be further investigated.
6. There is evidence that similar to Th17 cells Tc17 cells are regulated by Ca2+ and NFAT. Furthermore, they are important for the Th17-mediated pathogenicity of EAE (Huber et al., 2013). Due to Cd4-cre, also CD8+ T cells have ablated NFATc1. Therefore this population has to be analyzed in parallel. Please, The most weakness of the paper regards Th1 phenotype and IFNg production; in fact IFNg reduction observed in supernatants of MOG-restimulated splenic cells (both total and CD4+ T cells) and also in T cells derived from brain and spinal cord, is in contrast with data shown at the beginning of the paper, indicated Answer: The reviewer is right. We have now discussed possible environmental factors leading to differences between in vivo and in vitro.
Moreover the paper needs to be improved answering the following questions.
Major point: 1. Could be the reduced proliferation and cytokines production of MOG-specific splenic cells, related to the low amount of IL-2 produced by the same cells? Will be this proliferation and cytokine restored if rcIL-2 will be added to MOG-specific proliferation assay?
Answer: We cultured spleen cells from EAE mice at day 30 after EAE induction with MOG-peptide together with IL2. The proliferation of the cells cultured with MOG+IL2 showed always the same proliferation rate independent of the MOG dosis. The proliferation of NFATc1-deficient spleen cells was thereby reduced as compared to control cells. The cytokine levels of IFNg, IL17A and IL6 in the supernatants of these cells was reduced in the absence of NFATc1 whereas other analyzed cytokines were unchanged ( Figure 5).
2. What is the effect in terms of proliferation rate and cytokines production if NFATc1-deficient splenic cells will be TCR policlonally stimulated instead of antigen specific stimulation?
Answer: We cultured MOG-specific spleen cells from EAE mice at day 30 after EAE induction and This work presents a novel role for transcription factor NFATc1 in Th17 cells, using a T-cell conditional KO for NFATc1. NFATc1-deficient CD4 T cells polarized under Th717 conditions have less Th17 cytokines and less RORgt expression. An in vivo model was performed to demonstrate further this function. An EAE experiment was performed and analyzed quite completely, also analyzing data from brain and spinal cord samples. Their results also point out that IFNg expression is also altered by NFATc1 in this mouse model. Altogether, they nicely conclude that NFATc1 induces Th17 differentiation.

Comments:
In the Abstract, Introduction and Results it is mentioned that NFATc1 flox/flox CD4-Cre+ mice show NFATc1-deficiency in CD4-expressing cells. CD4 is expressed at the DP stage, which means that the Cre recombinase will be expressed FROM the DP stage, deleting NFATc1 since some point after that and in both CD4 and CD8 single-positive and mature T cells. Not only CD4 cells will be deficient in NFATc1, but also CD8 cells. At least deletion in both subsets has been seen in most gene-deletions by CD4-Cre. If these authors really only see deletion of NFATc1 in CD4 T cells, they should show a WB detecting NFATc1 in CD8 cells. If not, the writing should be changed to reflect deletion in both CD4 and CD8 T cells. √ Answer: The reviewer is right. NFATc1 is deleted in CD4+ as well as CD8+ in these mice. We also detected the reduction of NFATc1 expression in CD8+ T cells in the analyzed mice. Now we have changed the manuscript adding the point that NFATc1 is also deleted in CD8+ T cells in these mice. Fig. S2C demonstrate that, in vitro, there is a reduction in IFNg protein expression but not in RNA. They state that they "did not find any significant differences" but they should comment on the protein result, that does show a decrease (page 6, line 27).

Results shown in Suppl
NFATc1-deficient Th1 cells. We have now integrated that point in the discussion.
In the Discussion authors comment on the potential binding sites they see on the promoters of the cytokine genes but do not show any ChIP data to indicate their importance. However, they do not make any comment on RORgt, which is the master regulator, and which also shows to be affected by deletion of NFATc1. This should be rectified.
Answer: The reviewer is right. The potential binding sides for NFATc1 of promoters of several genes were described in publications from other groups. We were not able to find a publication showing that especially oter. Your manuscript has been re-reviewed and the comments of the referees are included at the bottom of this letter. Although the referees have recommended publication, some revisions to your manuscript have been requested. Therefore, I invite you to respond to the comments of the referees and revise your manuscript accordingly.
You should also pay close attention to the editorial comments included below. **In particular, please edit the language of your manuscript, as noted by referee #1, and your figure legends to follow Journal standards as outlined in the editorial comments. Failure to do this will result in delays in the re-review process.** If the revision of the paper is expected to take more than three months, please inform the editorial office.
Revisions taking longer than six months may be assessed by new referee(s) to ensure the relevance and timeliness of the data.
Once again, thank you for submitting your manuscript to European Journal of Immunology. We look forward to receiving your revision. Please change your wrong sentence in the Introduction: "In the promoter of NFATc1, binding sites for Il17a but not Il17f were found [14]". to the following: "Within the 17a promoter, but not the Il17f promoter, binding sites for NFAT were found [14]". Please avoid the term "deletion" when you mean inactivation of the Nfatc1 gene, or NFATc1 ablation. The term deletion stays for the "complete" deletion of gene.
It would be helpful when the entire MS could be carefully polished, both with respect to the English, typing errors and other small mistake.

Reviewer: 2
Comments to the Author The reviewed version of the paper appears greatly improved.
Please take in care the following suggestions: -It is not reported in the text the reference of Fig 2E -Pag 10 line 18, "data not shown" may be better than "unpublished data" -In case of flow cytometric evaluation you have to indicate "frequency " of positive cells instead of "number" of cells Reviewer: 3 Comments to the Author The authors have responded to our comments and the paper is ready to be accepted.

Second Revision -authors' response -28 January 2015
Reviewer: 1 Comments to the Author Please go again carefully through the entire MS and correct a number of flaws and errors. I want to show you only two of which that I found in the Introduction of your MS: Please change your wrong sentence in the Introduction: "In the promoter of NFATc1, binding sites for Il17a but not Il17f were found [14]". to the following: "Within the 17a promoter, but not the Il17f promoter, binding sites for NFAT were found [14]". Please avoid the term "deletion" when you mean inactivation of the Nfatc1 gene, or NFATc1 ablation. The term deletion stays for the "complete" deletion of gene.
It would be helpful when the entire MS could be carefully polished, both with respect to the English, typing errors and other small mistake.
Answer: We have corrected the manuscript as suggested by the reviewer.

Reviewer: 2
Comments to the Author The reviewed version of the paper appears greatly improved.
Please take in care the following suggestions: -It is not reported in the text the reference of Fig 2E -Pag 10 line 18, "data not shown" may be better than "unpublished data" -In case of flow cytometric evaluation you have to indicate "frequency " of positive cells instead of "number" of cells