Fungal sensing by dectin-1 directs the non-pathogenic polarization of TH17 cells through balanced type I IFN responses in human DCs

The non-pathogenic TH17 subset of helper T cells clears fungal infections, whereas pathogenic TH17 cells cause inflammation and tissue damage; however, the mechanisms controlling these distinct responses remain unclear. Here we found that fungi sensing by the C-type lectin dectin-1 in human dendritic cells (DCs) directed the polarization of non-pathogenic TH17 cells. Dectin-1 signaling triggered transient and intermediate expression of interferon (IFN)-β in DCs, which was mediated by the opposed activities of transcription factors IRF1 and IRF5. IFN-β-induced signaling led to integrin αvβ8 expression directly and to the release of the active form of the cytokine transforming growth factor (TGF)-β indirectly. Uncontrolled IFN-β responses as a result of IRF1 deficiency induced high expression of the IFN-stimulated gene BST2 in DCs and restrained TGF-β activation. Active TGF-β was required for polarization of non-pathogenic TH17 cells, whereas pathogenic TH17 cells developed in the absence of active TGF-β. Thus, dectin-1-mediated modulation of type I IFN responses allowed TGF-β activation and non-pathogenic TH17 cell development during fungal infections in humans.

Thank you for the opportunity to review your work. Major findings of the study: − The authors propose a model in which Dectin-1 controls the development of non-pathogenic (defined as IL-17 only producing) vs pathogenic (defined as IL-17 plus IFNg-double producing) Th17 cells via the amount of IFN-b induced in DCs. Low amounts of IFN-b and excess amounts of IFN-b would promote the generation of pathogenic Th17 cells at the expense of nonpathogenic Th17 cells. − The graded production of IFN-b depended on the ratio of IRF1 vs IRF5 nuclear translocation in DCs: while IRF5 promoted IFN-b expression, IRF1 inhibited it. Dectin-1 induced IFN-b, which in turn controlled the release of TGF-b by DCs by inducing the expression of the integrin ανβ8 that primed the latency-associated peptide for cleavage by MMP14, and thus the release of active TGF-b. Exogenous TGF-b could rescue the disruption of the IFN-b-driven release of TGF-b in terms of non-pathogenic Th17 differentiation in a DC-TC co-culture system. − While TGF-b was released by DCs through Dectin-1/IFN-b-mediated induction of ανβ8 integrin, high concentrations of IFN-b (which were facilitated by enhanced IRF5 action through inhibition of IRF1) induced BST2, an inhibitor of MMP14, which prevented TGF-b release by DCs.
General critique: This is a study that is fully conducted with human DCs and T cells. The study introduces the concept that DCs translate the graded induction of IFN-b by pattern recognition receptor ligands (in particular dectin-1) into differential amounts of TGF-b released in its active form. While low and high amounts of IFN-b that act back on DCs in an autocrine fashion would prevent the release of TGF-b through insufficient induction of ανβ8 integrin and inhibition of MMP14, respectively, medium amounts of IFN-b would facilitate the release of TGF-b. Sufficient availability of TGF-b would be necessary (and sufficient) to skew memory T cells into "non-pathogenic" Th17 cells (more IL-17 single producers than IL-17/IFN-g double producers). This concept provides a TGF-b centered view on Th17 skewing in the context of a relevant danger signal in fungal host defence. Some fundamental findings (IRF1 vs IRF5 mediated IFNb induction, antagonism of downstream pathways of IFN-b, i. e. integrin aνb8 vs BST2 induction) are brought into a comprehensive theory here. While this is all pretty attractive, some conceptual problems remain that are testable in the experimental set-up that the authors decided to use for their study (see specific comments).
Specific comments: 1. The concept of pathogenic vs non-pathogenic Th17 cells is still a matter of debate since it has not yet been decided whether these are rather states of Th17 cells than traits. Therefore, it might make a difference whether cytokine phenotypes  are tested in T cell cultures starting with naive T cells vs T cell cultures where T cells that already have been committed are tested (CD45RO positive). Key experiments should be repeated with naive T cells to see whether the concept of dectin-1 dependent graded expression of IFN-b also controls the "priming" (initial commitment) of T cells to IL-17 only producers vs IL-17/IFNg double producers. 2. The present study is difficult to reconcile with the results published by Zielinski et al. 2012(Zielinski et al., 2012 who propose that C. albicans would promote IL-17/IFN-g double producers (due to IL-1 induction in their co-culture system) while S. aureus would induce nonpathogenic Th17 cells that lack IFN-g production but co-produce IL-10. However, the coculture systems in both studies are quite different. While in that previous study irradiated autologous monocytes were used as APCs, GM-CSF/IL-4 induced DCs are used in the present study. Furthermore, Zielinski looked primarily at the "priming" of T cells and put "naive" T cells (CD45RO-) in co-culture, in the present study memory T cells (CD4+CD45RO+) were used. In addition, while Zielinski investigated the pathogen specific T cells (combining C. albicans as a source of antigens and adjuvants), the present study applies an oligoclonal stimulation system (SEB) in combination with curdlan or heat-killed C. albicans. Again, key experiments should be repeated in this prior set-up in order to show the robustness of the authors' concept. 3. IFN-b (in excess) curtailed MMP14 activity and thus prevented TGF-b release by DCs (resulting in loss of non-pathogenic Th17 cells in favor of pathogenic Th17 cells). While the BST2 mediated inhibition of MMP14 is convincing, the net effect of this pathway is confusing. It is not sufficiently clear how IFN-b (which promotes the production of IL-27 by myeloid cells) could still enhance pathogenic Th17 cells when IL-27 has been shown to dampen Th17 responses and most importantly, is a strong inducer of IL-10 in T cells (Guo et al., 2008;Stumhofer et al., 2007). What about IL-10 in the "excess" IFN-b condition? Other transcription factors (except c-MAF) might drive IL-10 here (such as Blimp-1 (encoded by PRMD1)).
Minor comments: p. 14, l. 331: wrong Reviewer #2 (Remarks to the Author): General comment: In this manuscript, the authors report that a regulated type I IFN production following dectin-1 stimulation is responsible for the generation of non-pathogenic (i.e., non-IFN-g-producing) Th17 cells. The mechanism underlying this phenomenon is dependent on the two IFNresponsive genes b8 (subunit of the avb8 integrin) and BST2. The avb8 integrin is essential for generation of active TGFb, which is the polarizing factor leading to non-pathogenic Th17 cells.
On the other hand, BST2 is induced following high levels of type I IFN and contributes (through MMP14) to the degradation of active TGFb and thus to the generation of pathogenic (i.e., co-expressing IFN-g) Th17. Therefore, dectin-1 fine-tunes type I IFN production to ensure enough active TGFb and the consequent development of non-pathogenic Th17 cells.
Overall, the Authors are to be commended for dissecting in depth the molecular mechanism underlying their observation. Experiments are in general technically sound, with proper controls, and the results clear and easy to interpret. However, the pathophysiological relevance of the Authors' findings is less clear to this reviewer. What would be the in vivo setting whereby excessive type I interferon leads to pathogenic Th17 development? Is this related to fungal infection or (absence of) dectin-1 signaling?
Specific comments: • Why were the co-cultures of DC performed with memory (rather than naïve) CD4+ T cells? Wouldn't this reflect plasticity rather than polarization? • The expression of the master transcriptional regulators for Th1 and Th17 (T-bet and Rorg-t, respectively) should be shown. • In all settings in which pathogenic Th17 were increased, Th1 cells were also significantly increased. This could lead to the conclusion that the effect of regulated type I IFN is on IFN-g production (regardless of the T helper subset). Do the authors have a hypothesis for this increase in IFN-g-producing cells?
• Is IFN-a also induced in this system (in addition to IFN-b)? • Fig S1A: has LPS been used as an alternative stimulus here and if so, why weren't ISGs induced?
Reviewer #3 (Remarks to the Author): Summary of the key results ok Originality and significance: if not novel, please include reference see report Data & methodology: validity of approach, quality of data, quality of presentation see report Appropriate use of statistics and treatment of uncertainties ok Conclusions: robustness, validity, reliability see report Suggested improvements: experiments, data for possible revision the T cell analyses requires significant additional experiments as indicated below References: appropriate credit to previous work? ok Clarity and context: lucidity of abstract/summary, appropriateness of abstract, introduction and conclusions all ok

Review
Gringhuis et at describe dectin-1 as a pathogen recognition receptor regulating the development of non-pathogenic Th17. They describe that dectin1 triggering by its ligands induces a type 1 IFN response which initiates the release of active TGFbeta and subsequently "non-pathogenic" Th17 cells, which are mainly defined by the absence of IFNg and a Th1 transcriptional profile whereas anti-inflammatory mediators, such as IL-10 and c-MAF are enhanced. Interestingly either blocking the type 1 IFNR or adding IFNbeta leads to induction of so called "pathogenic" Th17 cells coexpressing IFNg. The authors come to the conclusion that dectin-1 regulation of the IFNβ response at an intermediate level generates non-pathogenic TH17 differentiation and either too low or too high IFNβ results in pathogenic TH17 polarization. The manuscript has two main stories: 1. Dectin-1 signaling and regulation of active TGF-beta release 2. The effect of IFN/TGFbeta on human memory T cell differentiation. In my view the first point regarding transcriptional regulators downstream of dectin-1 in DC, leading to type IFN production and the biochemistry of active TGfbeta release have been nicely analyzed and the data look convincing to me, although this is clearly not my expertise and I cannot judge what is really new, since the pathways analyzed were all described before although maybe not in the same cellular context. Although the induction of IFN by dectin1 is known (e.g. Fresno et al Immunity 38, 1176Immunity 38, -1186Immunity 38, (2013), the fine-tuned IFN induction by dectin1 is a new concept in my view. However, some questions remain regarding this dose effect of dectin-1 signal and IFN (see below).
The second and in my view not well characterized part is that the fine balance of type I IFN decides over pathogenic or non-pathogenic Th17 responses and its molecular regulation via TGF-beta. Here I have several major concerns about the validity of the data and the experimental systems.
1. Pathogenic and non-pathogenic Th17 cells: The role of TGF-beta for non-pathogenic Th17 is known and even led to the term pathogenic versus non-pathogenic Th17. However, this has never been firmly established especially in humans. E.g. I am not aware that strong pathogenspecific Th17 responses in humans e.g. against Candida or S . aureus recapitulate such pathogenic or non-pathogenic phenotypes as described in the original publication by Zielinski et al Nature 2012. There Candida specific responses were claimed to induce pathogenic Th17 cells co-expressing IFNg whereas S. aureus induces non-pathogenic Th17 cell producing IL-10 which is actually in contrast to the data from Gringhuis et al. In my view protective or pathogenic function of a T cell subsets cannot be defined without the context of antigenspecificity., i.e. the same subset may have divergent function when targeting different pathogens. Therefore I would avoid overuse of the terms pathogenic/non pathogenic but stick to the actual facts, i.e. the induction of IFNg and other alterations of the transcriptional profile, which are actually not restricted to the Th17 cells in the cultures (see below). Also the term differentiation should be used for stable alterations which occur especially in naïve T cells, whereas transient "modulation" of gene expression may better describe the analyses in the manuscript.
2. The T cell stimulation system in general is unclear to me. a. The main effects on DC throughout the paper occur during the first hours of stimulation. However, all coculture experiments are done after 48 hours of stimulation. I am wondering what signals DC can still provide at this late time point. b. The assay also relies on a rather long in vitro expansion of memory T cells, which may introduce another strong experimental bias and leaves completely unclear which cells are targets or may simply survive under these conditions. The effect of DC modulation should be directly visible during the early interaction with DCs within hours or a few days. c. The memory cells used for coculture are quite undefined making it unclear which subsets are affected. It would be required to use defined starting populations: sorted Th1, Th17, Th17*(Sallusto definition), Th2 and naïve T cells to define which cell types are modulated (e.g. subsets defined by chemokine receptor patterns) or whether we talk about differentiation of naïve T cells. d. Overall it appears to me that the different conditions mainly modulate the Th1 profile of the culture, rather than inducing IFNg (or pathogenic phenotype) in a certain memory subset, such as Th17 cells. With TGFbeta repressing Th1 signatures but enhancing a "non pathogenic" signature, c-MAF, IL-10, which is well known and not restricted to Th17 cells. So we may look on two overlapping effects here which has to be addressed. e. It remains unclear what induces/recalls actually the IL-17 production in this culture system since it is not blocked by any of the tested factors.
Specific points: Fig.1 Dosage effect: The authors provide the view that dectin-1 signals always lead to the ideal IFN/TGFbeta levels. Is there no dose effect on dectin-1 signaling? Also, if the authors believe that high and low concentrations of IFN induce TGF-beta and subsequently IFN-g coexpressing Th17 cells and repress IL-10/c-MAF they should show it, e.g. IFN titration with DC matured in the absence of fungal stimulation. There are two effects of the co-culture: strong IL-17 induction or outgrowth of th17 cells but also massive reduction of IFNg (70 to 20%) in all cells. With anti IFNR and addition IFN there is less decrease of IFNg. Th17 are not affected.
-What induces IL-17 -The claim that IFNg/Th17 are specifically induced is not clear. The culture system is very undefined (total memory, 14 days), so it completely unclear which cells are modulated or survive. Also they should not call this Th17 differentiation since they start with memory cells. They should take either naïve T cells, sorted Th17 and Th17* cells (Sallusto protocol) and use Candida specific T cell directly.
-Much shorter stimulation times seem to be relevant: why 14 days? Is this physiologic? Modulation of memory cells/effect by short lived DC should be visible immediately (hours/few days) after stimulation.
-Also the effects are not consistent, while Candida induces Th17 (not sensitive to aIFNR) and reduces massively IFNg (restored by anti IFNR) addition of IFN reduces IL-17 and increases IFNg.
-Although under both conditions the relative frequency of IL17/IFNg double pos is indeed higher it is completely unclear what happens especially in quantitative terms (induction or selective survival). The authors should report about the quality of the culture, survival, expansion rates. Under both conditions (antiIFNR & IFNbeta) IFNg is massively induced which explains the increase in IL17/IFNg double pos.
-If this is a dosing effect of type 1 IFN (low dose: no effect on IL17 strong block of IFNghigh dose: block of IL-17 + induction of IFNg?) they should show it.
-IN 2C/D they focus on IL-17/IFNg: other cytokines should be included such as IL-10, IL-22, GMCSF or transcription factors such as c-MAF which are all analysed below on the transcriptional level. -The clear link to the role of type 1 IFN is missing and therefore the relevance of the data is unclear to me: o Remarkable RNA levels of IFNg, but only two CD patients were analysed o FACS stainings should be shown to confirm RNA analysis where possible, especially the critical ones: IL-10, IFNg, c-MAF, GMCSF, … o Are there for example more IFNg/IL-17 producers within candida albicans specific T cells (maybe data from literature).
Fig 2G/H: RNA analysis of in vitro cultured total IL-17 or sorted IFNG+ IL-17+ or IFNg-IL-17+ cells -Sorted IFNg+/-: results confirm expression profile e.g. enriched "pathogenic" genes in IFNg+ and non pathogenic genes in IFNg-. Which is a nice technical control but not surprising since they were sorted for IFNg.
-No difference between cells cultured with and without Curdlan/Candida, suggesting that the in vitro conditions alter the balance between the cells but not the cells per se (again indicating the critical role of the culture conditions as discussed for 2 A/B).
-Since the main effect of the antiIFNR and IFNbeta is actually on IFNg production, I am missing the single IL-17 negative cells and/or IFNg pos cells as a control, i.e. are TH17/IFNg cell uniqe or just reflecting a mixture between IFNg+ and IL-17+. -These markers should be confirmed by flow cytometry, in addition cytokine production in the SN should be integrated to support the overall effect on cytokine balance. -Again I am missing the IFNg only and the T cell culture without Curdlan/Candida modulated DCs as a control such as in Fig 2A/B since they have already huge IFNg production, Fig4: IFNR induces IL-27 (IL1, 6, 23 not affected by aIFNR) but this has no functional impact.
-Are there any high controls for the functionality of the anti IL-27 and blocking antibodies, since IL-27 has been described as a T cell modulator, e.g. to induce IL-10 production and Tr1 differentiation. This should be included.

Fig5
: Dectin1/IFN induces release of active TGFbeta Curd/Cand induces release of active TGbeta, blocked by aIFNR and anti dectin1 IFN blocks TGFbeta release in a dose dependent fashion and IRF1 or IRF5 knockdown in DC blocks active TGFbeta release. Claim: dectin1 generates optimal IFN whereas high and low dose IFN prevent the release of active TGF-beta: -No direct evidence for such a dose effect of IFN, this has to be shown as indicated above by titration of IFN in the absence of Curd/ Cand   Fig 5E,F,G TGF beta effect on "pathogenic" Th17 development?
-Again as seen in Fig 2 blockade of TGbeta, just like aIFNR, mainly leads to a drastic increase in IFNg but not restricted to Th17 cells. Therefore the increase in double positive IFNg/IL17 is rather a consequence of IFNg increase. Addition of TGFbeta has no direct effect but blocks the aIFNR induced IFNg induction, suggesting that TGFbeta in the culture released by Curd/Cand inhibits IFN-g production, as has been suggested before. Overall this does not suggest specific conversion into IFNg+/IL-17+ T cells by TGFbeta.
Fig5H mRNA profile of TGFbeta modulated Th17 -The mRNA supports in my view that TGFbeta mainly suppresses the overall Th1 signature and increases anti-inflammatory signature IL-10/cMAF without specifically affecting Th17 cells. That TGFbeta has this activity on IL-10 is already known.
Although we cannot offer to publish your paper in Nature Immunology, the work may be appropriate for another journal in the Nature Research portfolio. If you wish to explore suitable journals and transfer your manuscript to a journal of your choice, please use our <a href="https://mts-ni.nature.com/cgibin/main.plex?el=A6E4OMB1A7LFV3X3A9ftdhzKquxpaeHTr79FPtucQwZ">manuscript transfer portal</a>. If you transfer to Nature-branded journals or to the Communications journals, you will not have to re-supply manuscript metadata and files. This link can only be used once and remains active until used.
All Nature Research journals are editorially independent, and the decision to consider your manuscript will be taken by their own editorial staff. For more information, please see our <a href="http://www.nature.com/authors/author_resources/transfer_manuscripts.html?WT.mc_id =EMI_NPG_1511_AUTHORTRANSF&WT.ec_id=AUTHOR">manuscript transfer FAQ</a> page. Note that any decision to opt in to In Review at the original journal is not sent to the receiving journal on transfer. You can opt in to <i><a href="https://www.nature.com/natureresearch/for-authors/in-review">In Review</a></i> at receiving journals that support this service by choosing to modify your manuscript on transfer. In Review is available for primary research manuscript types only.

Author Rebuttal to Initial comments
Response to Reviewer #1

1.
The concept of pathogenic vs non-pathogenic Th17 cells is still a matter of debate since it has not yet been decided whether these are rather states of Th17 cells than traits. Therefore, it might make a difference whether cytokine phenotypes  are tested in T cell cultures starting with naive T cells vs T cell cultures where T cells that already have been committed are tested (CD45RO positive). Key experiments should be repeated with naive T cells to see whether the concept of dectin-1 dependent graded expression of IFN-b also controls the "priming" (initial commitment) of T cells to IL-17 only producers vs IL-17/IFNg double producers.
We thank the reviewer for the comment. As requested, we have repeated key TH outgrowth experiments using naive T cells. These new data support our findings and confirm that the dectin-1dependent graded expression of IFNβ critically determines the phenotype of the in vitro differentiated TH17 cells: limited IFNβ expression controls the development of T cells that are mainly IL-17 only producers, whereas blocking of IFNR signaling or excess IFNβ leads primarily to IL-17/IFNγ double producers. We have included these new data in Fig. 2, Supplementary Fig. 2 and Supplementary Fig. 5 and discussed the findings in the manuscript.

2.
The present study is difficult to reconcile with the results published by Zielinski et al. 2012(Zielinski et al., 2012 who propose that C. albicans would promote IL-17/IFN-g double producers (due to IL-1 induction in their co-culture system) while S. aureus would induce non-pathogenic Th17 cells that lack IFN-g production but co-produce IL-10. However, the coculture systems in both studies are quite different. While in that previous study irradiated autologous monocytes were used as APCs, GM-CSF/IL-4 induced DCs are used in the present study. Furthermore, Zielinski looked primarily at the "priming" of T cells and put "naive" T cells (CD45RO-) in coculture, in the present study memory T cells (CD4+CD45RO+) were used. In addition, while Zielinski investigated the pathogen specific T cells (combining C. albicans as a source of antigens and adjuvants), the present study applies an oligoclonal stimulation system (SEB) in combination with curdlan or heat-killed C. albicans. Again, key experiments should be repeated in this prior set-up in order to show the robustness of the authors' concept.
We have repeated key TH outgrowth experiments using naive T cells (see comment 1 above) in the absence of SEB and observe similar results as obtained with memory T cells in the presence of SEB, strongly suggesting that neither the use of naive vs memory T cells nor antigen specificity variances account for the observed differences.
However, another major difference between the setup of the experiments -which we had not previously realized -might explain the observed differences and would actually unite our results: while we primed our DCs for 48 h before co-culture with either naive or memory T cells, Zielinski et al. stimulated their APCs for only 3 h prior to addition of naive T cells. In our case, this is a deliberate time frame as it should closely resemble the physiological time frame in which activated DCs will transfer their message to the CD4 T cells, as DCs require 24-72 h to reach lymph nodes once they have entered the lymphatic system . This time period will also allow the DCs to develop a cytokine expression profile so they are appropriately equipped to deliver their message to T cells: our results show that it takes at least 8 h before αvβ8 integrin is present on DCs after recognition of ligands by dectin-1 and thus able to generate active TGFβ. This would mean that in the experiments of Zielinski et al., after only 3 h APC stimulation, naive T cells were subjected to differentiation signals in the absence of active TGFβ, which would explain the development of pathogenic TH17 cells. To test this hypothesis, we have performed experiments with 3 h curdlan-or C. albicans-primed DCs and naive T cells and we observed that the majority of the differentiated TH17 cells have a pathogenic phenotype, i.e. are IL-17/IFNγ double producers. The addition of rhTGFβ during co-culture resulted in the development of non-pathogenic TH17 cells (Reviewer Fig. 1-1). We would like to point out that another study by Bacher et al. showed that C. albicans-specific circulating memory TH17 cells in healthy persons have a nonpathogenic phenotype, in line with the results of our study . We have now discussed this in the manuscript and if requested we can include the data presented in the reviewer figure in the manuscript in order to reconcile our data with the study by Zielinski et al. 3. IFN-b (in excess) curtailed MMP14 activity and thus prevented TGF-b release by DCs (resulting in loss of non-pathogenic Th17 cells in favor of pathogenic Th17 cells). While the BST2 mediated inhibition of MMP14 is convincing, the net effect of this pathway is confusing. It is not sufficiently clear how IFN-b (which promotes the production of IL-27 by myeloid cells) could still enhance pathogenic Th17 cells when IL-27 has been shown to dampen Th17 responses and most importantly, is a strong inducer of IL-10 in T cells (Guo et al., 2008;Stumhofer et al., 2007). What about IL-10 in the "excess" IFN-b condition? Other transcription factors (except c-MAF) might drive IL-10 here (such as Blimp-1 (encoded by PRMD1)).
We thank the reviewer for noting this interesting observation. We observe that in the 'excess' IFNβ condition -which indeed results in excess IL-27 expression (we have now included these data in Fig.  4b) -expression of IL-10, and also transcriptional regulator c-Maf, is suppressed in the outgrown TH17 cells ( Fig. 2i and Supplementary Fig. 2d), suggesting that IL-27 is not the only factor controlling IL10 transcription in these T cells. IFNγ is a known suppressor of IL10 transcription . As the 'excess' IFNβ condition strongly induced IFNγ expression in the outgrown TH17 cells, we hypothesize that IFNγ counteracts the effects of IL-27R signaling on IL10 transcription. We have clarified this in the manuscript.
p. 15,l. 357: in the Fig. 7,panel d is missing. In the text, the description refers to Fig. 7e (not 7d, which is missing), and Fig. 7e (ll. 359,360,388) refers to the actual panel Fig. 7f.
We thank the reviewer for pointing out these errors and apologize that we did not triple check the figure references before submitting. Fig. 1-1 | a,b, Flow cytometry analyses of TH polarization by staining for intracellular IL-17 and IFNγ expression (FI, fluorescence intensity) in restimulated T cells, outgrown in vitro by coculture of naive CD4 + T cells with immature DCs (iDC) or DCs primed for 3 h (a,b) or 48 h (b) with curdlan or C. albicans CBS2712, in the absence or presence of recombinant human (rh) TGFβ1 (n = 3). Representative dot plots for independent donors are shown, with % positive cells indicated in each quadrant. In (b), the % IFNγand % IFNγ + cells per IL-17 + T cells are shown. Data in (b) represent mean ± s.d. of independent donors.

Response to Reviewer #2:
Overall, the Authors are to be commended for dissecting in depth the molecular mechanism underlying their observation. Experiments are in general technically sound, with proper controls, and the results clear and easy to interpret. However, the pathophysiological relevance of the Authors' findings is less clear to this reviewer. What would be the in vivo setting whereby excessive type I interferon leads to pathogenic Th17 development? Is this related to fungal infection or (absence of) dectin-1 signaling?
We thank the reviewer for the compliments and interesting comment. Our study shows that dectin-1 signaling strictly controls type I IFN responses to ensure the development of nonpathogenic TH17 responses during C. albicans infection. This allows fungal infections to be cleared without tissue damage and excessive inflammation. Bacher et al. have previously shown that circulating C. albicansspecific TH17 cells in healthy donors indeed carry the nonpathogenic phenotype . In contrast, TH17-driven pathologies are observed with other pathogenic fungal species such as Fonsaceae and Malassezia species, which are associated with excessive skin inflammation. Thus, it is possible that these pathogens interfere with dectin-1 signaling and as such dectin-1-induced type I IFN responses. For example, we have previously shown that Fonsaceae species also trigger Mincle signaling, besides dectin-1, which leads to IRF1 degradation (Wevers et al. 2014). As IRF1 is involved in limiting type I IFN responses in response to dectin-1 triggering, our data suggest that the degradation of IRF1 might underlie the induction of pathogenic TH17 responses to Fonsaceae species as a result of excess IFNβ. Moreover, pathogenic TH17 responses induced by excess IFNβ might be important in autoimmune diseases such as Crohn's disease where there is a strong IFN signature and pathogenic TH17 responses (Andreou et al. 2020). Thus, our findings that type I IFN responses play an essential role in dictating pathogenic versus non-pathogenic TH17 responses has broader implications and might even be important in viral infections. We have clarified this in the manuscript.

Specific comments:
• Why were the co-cultures of DC performed with memory (rather than naïve) CD4+ T cells?
Wouldn't this reflect plasticity rather than polarization?
We agree with the reviewer that the TH outgrowth experiments using memory T cells reflect phenotypic plasticity rather than differentiation. We have now clarified this in the manuscript. Furthermore, we have now also included TH outgrowth assays using naive T cells. These new data support our findings and confirm that the dectin-1-dependent graded expression of IFNβ critically determines the phenotype of the in vitro differentiated TH17 cells: limited IFNβ expression controls the development of T cells that are mainly IL-17 only producers, whereas blocking of IFNR signaling or excess IFNβ leads primarily to IL-17/IFNγ double producers. We have included these new data in Fig. 2, Supplementary Fig. 2 and Supplementary Fig. 5 and discussed the findings in the manuscript.

• The expression of the master transcriptional regulators for Th1 and Th17 (T-bet and Rorg-t, respectively) should be shown.
As requested, we have included flow cytometry analyses that show the expression of RORγt and T-bet in the outgrown TH17 cells in Fig. 2m. These data confirm our transcriptional analyses.
• In all settings in which pathogenic Th17 were increased, Th1 cells were also significantly increased. This could lead to the conclusion that the effect of regulated type I IFN is on IFN-g production (regardless of the T helper subset). Do the authors have a hypothesis for this increase in IFN-g-producing cells?
We agree with the reviewer that the increase in pathogenic TH17 cells coincides with the increase in TH1 cells in our TH outgrowth assays using memory T cells. TGFβR signaling is known to block T-bet expression and hence IFNG transcription, while stimulating c-Maf expression and therefore IL10 transcription , Rutz, Noubade et al. 2011. Also, IFNγ expression in turn blocks IL10 transcription . As such it is not surprising, and even to be expected, that expression of these genes is affected in both TH17 and TH1 cells under the influence of (dys)regulated type I IFN responses and TGFβ activation by DCs. We have now clarified this in the manuscript.
Furthermore, we have now included TH outgrowth assays using naive T cells, in which we observe that the regulated type I IFN responses specifically control the phenotype of the in vitro differentiated TH17 cells, while hardly affecting TH1 cells; even during dysregulated type I IFN responses, the level of TH1 cell induction remains the same. These data indicate that the effects observed in the TH outgrowth assays using memory T cells reflect the prior presence of differentiated TH1 cells. We have discussed this in the manuscript.
• Is IFN-a also induced in this system (in addition to IFN-b)?
We thank the reviewer for this interesting question. No, IFNα is not induced when dectin-1 signaling is triggered by curdlan or C. albicans. This is due to the presence of IRF1 that prevents binding of IFNβinduced IRF7 to the IFNA promoter, similar as occurs on the IFNB promoter (Reviewer Fig. 2-1).
• Fig S1A: has LPS been used as an alternative stimulus here and if so, why weren't ISGs induced?
No, the LPS control was only used when measuring IFNB expression after 2 h. We agree with the reviewer that it was unclear this way that LPS was not included with the ISG expression at 6 h, for which we apologize. We have changed the figure for clarity. We have previously shown that indeed LPS induces ISG expression after 6 h ).
Reviewer Fig. 2 We agree with the reviewer that the concept of pathogenic and non-pathogenic TH17 cells is not as well established in humans as in mice. However, the presence of pathogenic and nonpathogenic TH17 in humans has been shown in various studies and pathogenic TH17 cells are observed in various diseases (Annunziato et al. 2007, Lazarevic and Glimcher 2011, Patel and Kuchroo 2015, Bsat et al. 2019, Kamali et al. 2019. Indeed, we have analyzed circulating TH17 cells isolated from Crohn's disease patients and observed a pathogenic phenotype. Furthermore, we have extended these analyses to healthy donors and established an overall phenotype of human pathogenic and non-pathogenic TH17 cells. These analyses enabled us to interpret the molecular signatures obtained from sorted IL-17 + cells in culture and label them as either 'pathogenic' or 'non-pathogenic' without relying on merely absence or presence of IFNγ expression. Moreover, with regard to C. albicans infections, Bacher et al. showed that C. albicans-specific circulating memory TH17 cells in healthy persons have a non-pathogenic phenotype, in line with the results of our study . As requested, we have limited the use of pathogenic and nonpathogenic TH17 cells and more often refer to IL-17 + IFNγ + and IL-17 + IL-10 + Th17 cells. We are not aware of any reports that show that IFNγ-expressing TH17 cells (beside Zielinski et al.) and/or subsequent tissue damage accompanies Candida albicans infections, of course in situations where no further underlying pathologies exist. In fact, we hypothesize that a major difference in the setup between the experiments of Zielinksi et al. and our experiments might explain the apparent discrepancies and would actually unite our results: while we prime our DCs for 48 h before co-culture with either naive or memory T cells, Zielinski et al. stimulate their APCs for only 3 h prior to addition of the naive T cells. As our results show that it takes at least 8 h before αvβ8 integrin is present on DCs after recognition of ligands by dectin-1 and thus able to generate active TGFβ, this would mean that in the experiments of Zielinski et al., after only 3 h APC stimulation, the naive T cells are subjected to differentiation/polarization signals in the absence of active TGFβ and thus elicit a pathogenic TH17 response. To test this hypothesis, we have performed experiments with 3 h curdlan-or C. albicans-primed DCs and naive T cells and we observed that the majority of the differentiated TH17 cells are IL-17/IFNγ double producers. The addition of rhTGFβ during co-culture resulted in the development of non-pathogenic TH17 cells (Reviewer Fig. 3-1). We have now discussed this in the manuscript and if requested we can include the data presented in the reviewer figure in the manuscript in order to reconcile our data with the study by Zielinski et al. Also the term differentiation should be used for stable alterations which occur especially in naïve T cells, whereas transient "modulation" of gene expression may better describe the analyses in the manuscript.
We agree with the reviewer that the experiments with memory T cells support the role of the defined mechanism in determining the phenotype of the outgrown TH17 cells, thereby reflecting phenotypic plasticity, i.e. 'modulation', rather than differentiation, i.e. stable alterations. We have now clarified this in the manuscript. Furthermore, we have now also included TH outgrowth assays using naive T cells.
These new data support our findings and confirm that the dectin-1-dependent graded expression of IFNβ and subsequent TGFβ activation critically determines the phenotype of the in vitro differentiated TH17 cells: limited IFNβ expression controls the development of T cells that are mainly IL-17 only producers, whereas blocking of IFNR signaling or excess IFNβ leads primarily to IL-17/IFNγ double producers. We have included these new data in  We determine transcriptional effects early after stimulation (2-6 h), however the presence of proteins like cytokines (e.g. active TGFβ) and ISGs (β8, BST2) -and also co-stimulatory molecules -is detected much later (24-48 h). We have chosen this later time point also as DC-T cell encounters in lymphoid tissues will only occur after DCs encounter pathogens at mucosal sites, after which DCs require 24-72 h to reach lymph nodes once they have entered the lymphatic system (de Winde et al. 2020). Therefore, we feel that 48 h poststimulation reflects a more physiological time frame that allows DCs to establish a protein expression profile so they are appropriately equipped to deliver their message to T cells. Moreover, our experiments with 3 h primed DCs as described above (see comment 1) and shown in Reviewer Figure 3-1 suggest that shorter time periods are not sufficient for the development of a full-sized protein expression profile.

b. The assay also relies on a rather long in vitro expansion of memory T cells, which may introduce another strong experimental bias and leaves completely unclear which cells are targets or may simply survive under these conditions. The effect of DC modulation should be directly visible during the early interaction with DCs within hours or a few days.
We respectfully disagree with the reviewer that the long expansion period might introduce experimental bias as we use the same assay for all conditions and any experimental bias that for example favored survival of a certain TH17 subtype would eclipse any differences between our various conditions. We have now included new data using naive T cells in an adapted TH outgrowth assay with a short 5 days coculture period, which recaptures our data obtained after prolonged memory T cell expansion. We have included these new data in Fig. 2, Supplementary Fig. 2 and Supplementary Fig. 5 and discussed the findings in the manuscript.

c.
The memory cells used for coculture are quite undefined making it unclear which subsets are affected. It would be required to use defined starting populations: sorted Th1, Th17, Th17*(Sallusto definition), Th2 and naïve T cells to define which cell types are modulated (e.g. subsets defined by chemokine receptor patterns) or whether we talk about differentiation of naïve T cells.
We agree with the reviewer that it is unclear what memory subsets are affected in the TH outgrowth experiments using memory T cells. Flow cytometry analyses show that the isolated memory T cells contained <1% naive T cells. However, the focus of our manuscript is on the regulation of the phenotypic traits of TH17 cells and as such we have not further investigated the origin of the outgrown TH17 cells.
Moreover, we have now included TH outgrowth assays using naive T cells and these new data confirm that the dectin-1-dependent regulated expression of IFNβ and subsequent TGFβ activation critically determines the phenotype of the in vitro differentiated TH17 cells. We have included these new data in

d.
Overall it appears to me that the different conditions mainly modulate the Th1 profile of the culture, rather than inducing IFNg (or pathogenic phenotype) in a certain memory subset, such as Th17 cells. With TGFbeta repressing Th1 signatures but enhancing a "non pathogenic" signature, c-MAF, IL-10, which is well known and not restricted to Th17 cells. So we may look on two overlapping effects here which has to be addressed.
The reviewer raises an interesting point. Indeed, as the reviewer mentions, it it is well known that TGFβR signaling blocks T-bet expression and hence IFNG transcription, while stimulating c-Maf expression and therefore IL10 transcription , while in turn IFNγ blocks IL10 transcription . As such it is not surprising, and even to be expected, that expression of these genes is affected in both TH17 and TH1 cells under the influence of type I IFN responses and TGFβ activation by DCs. We have now clarified this in the manuscript. Thus, we completely agree with the reviewer that the increase in pathogenic TH17 cells coincides with the increase in TH1 cells in our TH outgrowth assays using memory T cells. The focus of our manuscript is on the regulation of the phenotypic traits of TH17 cells and as such we have not further investigated the regulation of TH1 cells.
Importantly, we have now included TH outgrowth assays using naive T cells, in which we observe that the regulated type I IFN responses specifically control the phenotypic traits of TH17 cells, while hardly affecting TH1 cells. We have included these new data in Fig. 2, Supplementary Fig. 2 and Supplementary Fig. 5. These data indicate that the effects observed in the TH outgrowth assays using memory T cells reflect the prior presence of differentiated TH1 cells. Moreover, the effect of regulated type I IFN is not merely on IFNγ and IL-10 expression -via regulated TGFβR signaling -as during excess IFNβ conditions -that lead to excess IL-27 (we have now added those data in Fig. 4b) -IL-27R signaling interferes with IL-17 production in T cells ( Fig. 2 and Fig. 4). We have discussed this in the manuscript.

e. It remains unclear what induces/recalls actually the IL-17 production in this culture system since it is not blocked by any of the tested factors.
We and others have previously shown that IL-23 and IL-1β expression is required for IL-17 production in the TH outgrowth assay with memory T cells (van Beelen et al. 2007, Gringhuis et al. 2011. As the levels of IFNβ induced by dectin-1 triggering do not affect IL-23 and IL-1β expression (Fig. 4a), it would have been unexpected if IL-17 production had been blocked. We have clarified this in the manuscript. The ligands we have used in the current research are strong ligands of dectin-1 and there is no dose effect on dectin-1 signaling (Reviewer Fig. 3-2).

Also, if the authors believe that high and low concentrations of IFN induce TGF-beta and subsequently IFN-g coexpressing Th17 cells and repress IL-10/c-MAF they should show it, e.g. IFN titration with DC matured in the absence of fungal stimulation.
The reviewer raises an interesting question. However, either differentiation of naive T cells to TH17 cells or recall of IL-17 production in memory T cells requires dectin-1-induced cytokines such as IL-23 and IL-1β (see above, comment 2e). As such, treatment of DCs with IFNβ alone will not provide the signals necessary for induction of TH17 responses. This is also apparent in the experiments shown in Fig. 2b and Fig. 2f. Additionally, stimulation of DCs with IFNβ alone (concentration range) does not result in release of active TGFβ: only during co-stimulation with curdlan or C. albicans all required signals that lead to TGFβ activation are present (Fig. 5b). Thus, without dectin-1 triggering (either via fungal stimulation or curdlan) we are not able to mimic the effects of regulated IFNβ expression on TH17 polarization. Replacing dectin-1 triggering by a random PRR ligand to mature DCs would result in unpredictable expression patterns of both the TH17-inducing cytokines IL-23, IL-6 and IL-1β as well as IFNβ.

-What induces IL-17
Dectin-1-induced IL-23 and IL-1β expression is required for IL-17 production in the TH outgrowth assay with memory T cells (see comment 2e) (van Beelen et al. 2007, Gringhuis et al. 2011. As the levels of IFNβ induced by dectin-1 triggering do not affect IL-23 and IL-1β expression (Fig. 4a), IL-17 production in outgrown TH 17 cells is not affected.
The lower % of IFNγ-expressing cells in our TH outgrowth assays using memory T cells likely reflects the levels of TGFβ in the culture. As discussed above (see comment 2d), TGFβR signaling blocks T-bet expression and hence IFNG transcription, while stimulating cMaf expression and therefore IL10 transcription , while in turn IFNγ blocks IL10 transcription . As TGFβ activation is blocked in the presence of blocking IFNR antibodies as well as excess IFNβ, IFNG transcription is no longer blocked in both TH17 and TH1 cells, hence the observed 'less decrease'.
-The claim that IFNg/Th17 are specifically induced is not clear. The culture system is very undefined (total memory, 14 days), so it completely unclear which cells are modulated or survive. Also they should not call this Th17 differentiation since they start with memory cells. They should take either naïve T cells, sorted Th17 and Th17* cells (Sallusto protocol) and use Candida specific T cell directly.
We agree with the reviewer that the experiments with memory T cells support the role of the defined mechanism in determining the phenotype of the outgrown TH17 cells, thereby reflecting phenotypic plasticity, i.e. 'modulation', rather than differentiation, i.e. stable alterations. We have now clarified this in the manuscript.
Furthermore, we have now also included TH outgrowth assays using naive T cells with a short 5-days coculture period, which recaptures our data obtained after prolonged memory T cell expansion and confirms that the dectin-1-dependent graded expression of IFNβ and subsequent TGFβ activation critically determines the phenotype of the in vitro differentiated TH17 cells. In the TH outgrowth assays using naive T cells, we observe that the regulated type I IFN responses specifically control the phenotypic traits of TH17 cells, while hardly affecting TH1 cells. We have included these new data in Fig.  2, Supplementary Fig. 2 and Supplementary Fig. 5 and discussed the findings in the manuscript.
-Much shorter stimulation times seem to be relevant: why 14 days? Is this physiologic?

Modulation of memory cells/effect by short lived DC should be visible immediately (hours/few days) after stimulation.
We agree with the reviewer that effects of DC-induced protein expression profiles are visible after 4-5 days (not hours), as has been reported before. We have now included new data using naive T cells in an adapted TH outgrowth assay with a short 5 days co-culture period, which recaptures our data obtained after prolonged memory T cell expansion. The long expansion time of the memory T cells is a practical issue to obtain enough cells to perform our analyses. (Of note, we do not expand the memory T cells for a set time period of 14 days, but we keep them in culture until they reach a resting state, which is typically between 11-14 days after the start of the co-culture). The 5 days co-cultures with naive T cells are started with 10 times more T cells than the co-cultures with memory T cells to overcome this obstacle.
-Also the effects are not consistent, while Candida induces Th17 (not sensitive to aIFNR) and reduces massively IFNg (restored by anti IFNR) addition of IFN reduces IL-17 and increases IFNg.
We are afraid that the reviewer has misunderstood, and we will further clarify this in the manuscript. It is to be expected that the conditions in which IFNR signaling is blocked or excess IFNβ is present result in different outcomes as both conditions have distinct effects on the DC-derived cytokines (IL-23, IL-1β, IL-6, IL-27 and TGFβ) that affect IL-17 and IFNγ expression in T cells: (a) during the 'normal' situation with curdlan-or C. albicans-primed DCs, dectin-1 signaling induces IL-23, IL-1β and IL-6 expression (Fig. 4a), which induce IL-17 production in T cells. Simultaneously, dectin-1-induced graded type I IFN responses lead to TGFβ activation ( Fig. 5bd) and subsequent TGFβR signaling in differentiated T cells blocks T-bet expression and as such IFNγ expression. The type I IFN responses also lead to IL-27 expression (Fig. 1g, Fig. 4b), however these levels remain below the threshold levels that are required for IL-27 to block IL-17 production in T cells (Fig. 4c,d).
when IFNR signaling is blocked, this has no effect on IL-23, IL-1β and IL-6 expression ( Fig.  4a), while IL-27 expression is attenuated (Fig. 4b) and thus IL-17 production in T cells remains unaltered. Concurrently, the release of active TGFβ is blocked (Fig. 5b) as this depends on IFNR-induced αvβ8 expression ( Fig. 6a-h) -and as such TGFβR signaling can no longer block IFNγ expression in T cells.

(c)
during 'excess' IFNβ condition (either by addition of rhIFNβ or silencing of IRF5), IL-27 expression by DCs is increased (we have added those data to Fig. 4b), now reaching levels that do interfere with IL-17 production in T cells, as also seen in the 'excess' rhIL-27 condition (Fig. 4d). Concurrently, the release of active TGFβ is blocked (Fig. 5d) -as enhanced type I IFN responses also lead to increased levels of BST2 expression (Fig. 8b,e), which interferes with MMP14-mediated release of active TGFβ (Fig. 8i,j) -and as such TGFβR signaling can no longer block IFNγ expression in T cells, similar as seen during IFNR block (b).

-Although under both conditions the relative frequency of IL17/IFNg double pos is indeed higher it is completely unclear what happens especially in quantitative terms (induction or selective survival). The authors should report about the quality of the culture, survival, expansion rates. Under both conditions (antiIFNR & IFNbeta) IFNg is massively induced which explains the increase in IL17/IFNg double pos.
We have now included new data using naive T cells in an adapted TH outgrowth assay with a short 5 days co-culture period, which recaptures our data obtained after prolonged memory T cell expansion. These data confirm that the dectin-1-dependent regulated expression of IFNβ and subsequent TGFβ activation critically determines the phenotype of the in vitro differentiated TH17 cells and is not a result of selective cell survival or a bias in the culture system. Interestingly, in these TH outgrowth assays using naive T cells, we observe that the regulated type I IFN responses specifically control the phenotypic traits of TH17 cells, while hardly affecting TH1 cells. We have included these new data in Fig.  2, Supplementary Fig. 2 and Supplementary Fig. 5 and discussed the findings in the manuscript.
-If this is a dosing effect of type 1 IFN (low dose: no effect on IL17 strong block of IFNg -high dose: block of IL-17 + induction of IFNg?) they should show it.
As mentioned above (see 2nd specific comment), the reviewer raises an interesting question. However, either differentiation of naive T cells to TH17 cells or recall of IL-17 production in memory T cells requires dectin-1-induced cytokines such as IL-23 and IL-1β (see above, comment 2e). As such, treatment of DCs with IFNβ alone will not provide the signals necessary for induction of TH17 responses. This is also apparent in the experiments shown in Fig. 2b and Fig. 2f. Additionally, stimulation of DCs with IFNβ alone (concentration range) does not result in release of active TGFβ: only during co-stimulation with curdlan or C. albicans all required signals that lead to TGFβ activation are present (Fig. 5b). Thus, without dectin-1 triggering (either via fungal stimulation or curdlan) we are not able to mimic the effects of regulated IFNβ expression on TH17 polarization. Replacing dectin-1 triggering by a random PRR ligand to mature DCs would result in unpredictable expression patterns of both the TH17-inducing cytokines IL-23, IL-6 and IL-1β as well as IFNβ.
Instead, we mimic low and high IFNβ concentrations by other means: blocking IFNR antibodies ( Fig. 2l and Supplementary Fig. 2, Fig. 5b) and blocking αvβ8 antibodies (Fig. 6a, Fig. 6i and Supplementary Fig. 2) mimic a no/low IFNβ environment, resulting in no TGFβ activation by DCs and thus IFNγ induction in T cells, without affecting IL-17 production in T cells that is mediated by dectin-1-induced IL-23 and IL-1β, which are not affected by low IFNβ levels (Fig. 4a). On the other hand, excess IFNβ ( Fig. 2l and Supplementary Fig. 2, Fig. 5c) creates a high IFNβ environment that interferes with TGFβ activation by DCs (due to high BST2 levels) and thus IFNγ induction in T cells, while at the same time high levels of IFNβ -induced IL-27 interfere with IL-17 production in T cells. Excess IL-27 ( Fig. 4c-e and Supplementary Fig. 2) mimics the part of a high IFNβ environment that interferes with IL-17 induction in T cells, without interfering with TGFβ activation and thus IFNγ induction in T cells remains suppressed. Overexpression of BST2 (Fig. 8j, Fig. 8k and Supplementary  Fig. 2) mimics the part of a high IFNβ environment that results in no TGFβ activation by DCs and thus allows IFNγ induction in T cells, without affecting IL-17 production in T cells. As requested, we have added flow cytometry analyses that show the expression of transcription factors RORγt, T-bet and c-Maf, as well as other cytokines (IL-10 and GM-CSF) and IL-1R1 by the outgrown TH17 cells in Fig. 2m. We included the analyses of circulating TH17 cells isolated from Crohn's disease patients to set the 'baseline' for pathogenic TH17 cells so we could demonstrate the similarities with TH17 cells that developed in our in vitro TH outgrowth assays. We have clarified this in the manuscript.
o Remarkable RNA levels of IFNg, but only two CD patients were analysed For further confirmation, we have added data from two more CD patients. As these are diagnosed but yet untreated patients, they are hard to find.  The gene expression profile of human pathogenic and non-pathogenic TH17 cells is less well established than that of mice TH17 cells. Moreover, distinct markers that are linked to one of these phenotypes have been described in separate papers. We have therefore performed these analyses to establish the overall phenotype of human pathogenic and non-pathogenic TH17 cells. Moreover, these analyses allowed us to interpret the molecular signature obtained from overall TH17 cells of blood of patients/healthy donors and TH outgrowth cultures.

-No difference between cells cultured with and without Curdlan/Candida, suggesting that the in vitro conditions alter the balance between the cells but not the cells per se (again indicating the critical role of the culture conditions as discussed for 2 A/B).
We are unsure to what the reviewer is referring as we clearly show that memory T cells, and now also naive T cells, that are co-cultured in the presence of curdlan-or C. albicans-primed DCs demonstrate IL-17 production, while immature DCs do not induce this response.

-
Since the main effect of the antiIFNR and IFNbeta is actually on IFNg production, I am missing the single IL-17 negative cells and/or IFNg pos cells as a control, i.e. are TH17/IFNg cell uniqe or just reflecting a mixture between IFNg+ and IL-17+.
The IFNγ + TH17 cells have a unique phenotype and are not a mixture of IFNγ + and IL-17 + cells: all molecular analyses in Fig. 2l (previously Fig. 2h) -after IFNR block or excess IFNβ treatment of DCswere performed on sorted IL-17 + cells and as such do not contain single IFNγ + cells. As the focus of our manuscript is on the regulation of the phenotypic traits of TH17 cells, we have not further investigated the expression of the various genes in TH1 cells. Fig 2A/B on RNAlevel. -These markers should be confirmed by flow cytometry, in addition cytokine production in the SN should be integrated to support the overall effect on cytokine balance.

2H: Confirmation of IFNg/IL-17 results from
As requested, we have added flow cytometry analyses that show the expression of transcription factors RORγt, T-bet and c-Maf, cytokines IFNγ, IL-10 and GM-CSF as well as IL-1R1 by the outgrown TH17 cells in Fig. 2m.
We have also measured IL-17A and IFNγ in the supernatant: the ELISA data corroborate our flow cytometry analyses and demonstrate that only excess IFNβ levels reduce IL-17 production, while blocking IFNR or TGFβR signaling greatly enhances IFNγ production by T cells (Reviewer Fig. 3-3). If requested we can include the data presented in the reviewer figure in the manuscript.
-Again I am missing the IFNg only and the T cell culture without Curdlan/Candida modulated DCs as a control such as in Fig 2A/

B since they have already huge IFNg production,
The molecular analyses in Fig. 2l (previously Fig. 2h) -after IFNR block or excess IFNβ treatment of DCs -were performed on sorted IL-17 + cells and as such do not contain single IFNγ + cells. As the focus of our manuscript is on the regulation of the phenotypic traits of TH17 cells, we have not further investigated the expression of the various genes in TH1 cells. However, we have now included TH outgrowth assays using naive T cells, in which we observe that the regulated type I IFN responses specifically control the phenotypic traits of TH17 cells, while hardly affecting TH1 cells. In co-cultures of immature DCs with naive T cells, hardly any IFNγ-producing cells are induced, supporting an important role for DC maturation and the establishment of a full-sized cytokine expression profile. We have included these new data in Fig. 2, Supplementary Fig. 2 and Supplementary Fig. 5. These data indicate that the effects observed in the TH outgrowth assays using memory T cells reflect the prior presence of differentiated TH1 cells.

Fig4
: IFNR induces IL-27 (IL1, 6, 23 not affected by aIFNR) but this has no functional impact. -Are there any high controls for the functionality of the anti IL-27 and blocking antibodies, since IL-27 has been described as a T cell modulator, e.g. to induce IL-10 production and Tr1 differentiation. This should be included.
The antibody is functional as we have shown previously  As mentioned above (see 2nd specific comment), the reviewer raises an interesting question. However, either differentiation of naive T cells to TH17 cells or recall of IL-17 production in memory T cells requires dectin-1-induced cytokines such as IL-23 and IL-1β (see above, comment 2e). As such, treatment of DCs with IFNβ alone will not provide the signals necessary for induction of TH17 responses. This is also apparent in the experiments shown in Fig. 2b and Fig. 2f. Additionally, stimulation of DCs with IFNβ alone (concentration range) does not result in release of active TGFβ: only during co-stimulation with curdlan or C. albicans all required signals that lead to TGFβ activation are present (Fig. 5b). Thus, without dectin-1 triggering (either via fungal stimulation or curdlan) we are not able to mimic the effects of regulated IFNβ expression on TH17 polarization. Replacing dectin-1 triggering by a random PRR ligand to mature DCs would result in unpredictable expression patterns of both the TH17-inducing cytokines IL-23, IL-6 and IL-1β as well as IFNβ.
Instead, we mimic low and high IFNβ concentrations by other means: blocking IFNR antibodies ( Fig. 2l and Supplementary Fig. 2, Fig. 5b) and blocking αvβ8 antibodies (Fig. 6a, Fig. 6i and Supplementary Fig. 2) mimic a no/low IFNβ environment, resulting in no TGFβ activation by DCs and thus IFNγ induction in T cells, without affecting IL-17 production in T cells that is mediated by dectin-1-induced IL-23 and IL-1β, which are not affected by low IFNβ levels (Fig. 4a). On the other hand, excess IFNβ ( Fig. 2l and Supplementary Fig. 2, Fig. 5c) creates a high IFNβ environment that interferes with TGFβ activation by DCs (due to high BST2 levels) and thus IFNγ induction in T cells, while at the same time high levels of IFNβ-induced IL-27 interfere with IL-17 production in T cells. Excess IL-27 (Fig. 4c-e and Supplementary Fig. 2) mimics the part of a high IFNβ environment that interferes with IL-17 induction in T cells, without interfering with TGFβ activation and thus IFNγ induction in T cells remains suppressed. Overexpression of BST2 (Fig. 8j, Fig. 8k and Supplementary  Fig. 2) mimics the part of a high IFNβ environment that results in no TGFβ activation by DCs and thus allows IFNγ induction in T cells, without affecting IL-17 production in T cells. Indeed, as discussed above (see comment 2d), it it is well known that TGFβR signaling blocks T-bet expression and hence IFNG transcription, while stimulating c-Maf expression and therefore IL10 transcription . Also, IFNγ expression in turn blocks IL10 transcription . As such it is not surprising, and even to be expected, that expression of these genes is affected in both TH17 and TH1 cells under the influence of type I IFN responses and TGFβ activation by DCs. We have now clarified this in the manuscript. The focus of our manuscript is on how dectin-1 signaling specifically directs various processes to make sure that active TGFβ is released to ensure development of IL10-producing TH17 cells for fungal clearance and as such we have not further investigated the regulation of TH1 cells. Furthermore, we have now included TH outgrowth assays using naive T cells, in which we observe that the regulated type I IFN responses specifically control the phenotypic traits of TH17 cells, while hardly affecting TH1 cells. We have included these new data in Fig.  2, Supplementary Fig. 2 and Supplementary Fig. 5. These data indicate that the effects observed in the TH outgrowth assays using memory T cells reflect the prior presence of differentiated TH1 cells. Fig. 3-1 | a,b, Flow cytometry analyses of TH polarization by staining for intracellular IL-17 and IFNγ expression (FI, fluorescence intensity) in restimulated T cells, outgrown in vitro by coculture of naive CD4 + T cells with immature DCs (iDC) or DCs primed for 3 h (a,b) or 48 h (b) with curdlan or C. albicans CBS2712, in the absence or presence of recombinant human (rh) TGFβ1 (n = 3). Representative dot plots for independent donors are shown, with % positive cells indicated in each quadrant. In (b), the % IFNγand % IFNγ + cells per IL-17 + T cells are shown. Data in (b) represent mean ± s.d. of independent donors. Reviewer Fig. 3-2 | RT-PCR analyses of IFNB, IL6 and IL1B relative mRNA levels in DCs at 2 or 6 h after stimulation with a concentration range of curdlan (n = 3). Results from RT-PCR were normalized to the expression of reference household gene GAPDH and shown relative to 2 h curdlan (10 µg/ml) stimulation (IFNB) or 6 h curdlan (10 µg/ml) stimulation (IL6, IL1B). Data represent mean ± s.d. of independent donors.

General quality of the culture system: In my previous review I raised the point that it is difficult to understand the co-culture system the authors call "T cell outgrowth assay" (14 days, 2 day preactivation of DCs). The authors now show that DCs stimulated for 3h and 48h have different effects on T cells. This is ok and interesting. However, the authors still do not provide any data about the growth/survival of the T cells throughout the culture. It is well known that during culture small subsets may grow out especially if the overall proliferation is not homogenous. Thus standard parameters should be cell survival, expansion, viability allowing to estimate whether the cultures are comparable.
The new short term co-cultures of DCs with naïve T cells corroborate the results obtained with the long term co-cultures (T cell outgrowth assay). These data clearly show that short or long term assays are suitable to investigate the signals required for induction of pathogenic and nonpathogenic T cells by DCs.
We agree with the reviewer that during culture small subsets may grow out, but that is exactly the idea behind the DC-T cell co-cultures and it is therefore also called T cell outgrowth assay. In the DC-T cell co-cultures, only T cells that react to the alloantigens on DCs will survive and expand over 11-14 days allowing them to become the majority of the culture. During that time (but also as we have shown within 5 days) they develop their TH17 signature profile depending on the cues provided by the DCs. Thus, overall proliferation will indeed not be homogenous, and as such survival data will not add to the conclusions that can be drawn. When the signal from the mature DCs changes, so will the response of the T cells, meaning that cell survival/expansion/viability of the cultures will not be comparable, but reflect the change of the provided DC signal. This is particularly clear for our co-cultures in the presence of rhIFNβ or rhIL-27 that demonstrated significantly less T cell proliferation, which is exactly in line with our hypothesis and known literature.
We can provide the viability and cell survival if necessary but it will not add to the conclusions as there will be differences between conditions. We can clarify this in the discussion and also emphasize that the short term DC-T cell co-cultures corroborate the long term T cell outgrowth assay. These assays have been performed by various groups including Zielinski et al. and are considered the best assays to investigate differentiation cues.

I still disagree with the authors and would say that cytokine induction should be visible early, after a few days. Otherwise it would make biologically no sense.
We agree with the reviewer that cytokine induction is visible early, after a few days as we indeed observe a strong cytokine induction in the short 5 days time period of our DC-naive T cell cocultures (Fig. 2a,b and Supplementary Fig. 5). The data with the new short term DC-T cell cocultures corroborate our results obtained with the long term DC-memory T cell cultures. We can also perform a short term culture with memory T cells, but all reviewers seem to agree that the inclusion of the naïve T cell-DC co-cultures support our hypothesis. This is important to understand whether we really talk about induction of IFNg/IL-17 or just better survival, e.g. due to more costimulation by dectin1 matured DC. As previously mentioned, I don´t understand why human memory T cells activated for 14 days should not contain any Th17 cells (Fig 2 e,

f). Sallusto and colleagues showed nicely that Th17 and Th17*(IFNg) cells and other Th subsets can be directly sorted from blood according to chemokine receptor expression profile, and these cells produce a high amount of signature cytokines. I still think it is required to sort different memory subsets before coculture to distinguish between induction or survival.
We completely agree with the reviewer that TH17 cells can be directly sorted from blood -although we prefer using IL-17 stainings over chemokine receptor stainings. We used exactly that strategy to analyze IL-17+ CD4 T cells from blood of healthy persons and Crohn's disease patients (Fig.  2i,j). However, these cells constitute only a small fraction: we isolate ~ 100.000 IL-17+ cells from 15 million CD4+ T cells (this varies per donor), which is < 1%. These numbers are in line with literature [Simplified assay for enrichment of primed human Th17 and Tc17 lymphocytes from peripheral blood | Translational Medicine Communications | Full Text (biomedcentral.com)]. Obviously, during our memory T cell outgrowth assay, immature DCs do not provide the signals for T cells to proliferate and after re-stimulation only those <1% will produce IL-17, which is exactly what we see (Fig.  2e,f).
Instead of sorting different memory T cells, we have repeated the experiments as requested with naive T cells and we observe the same changes in differentiation profiles. These data support our hypothesis that IFN signaling controls TH17 differentiation. We would like to stress that the focus of the manuscript is not what cells are triggered to become IL-17-producing cells -although certainly an interesting research question -but that dectin-1 signaling specifically induces IL-17+IFNγ-cells. Therefore we believe that sorting different memory T cells and determine which subset is more efficiently induced into TH17 cells is beyond the scope of our manuscript.

Th17 induction Furthermore I still don´t understand which signals the 48h activated DC mediate to induce IL-17 (or alternatively: support better expansion of pre-existing Th17 cells). The authors write in the response letter that this has been previously been shown to depend on IL-1,-23 but then they should show that DCs after 48h still produce these cytokines and that they are relevant for the Th17 induction. By selectively blocking these cytokines and also IL-12 it can also be excluded that IFNg induction is not driven by IL-12 (as mentioned before to me it looks overall like strong induction of IFNg in all cells not only in the Th17 cells).
We have performed the suggested experiments in the past (see figure below), and if required we can repeat these experiments and include these in the manuscript.
As we have mentioned in the response letter, other groups have performed similar experiments with the same setup and same blocks, although the primary signal was then provided by NOD2 ligands, also showing that IL-1β and IL-23 are required for DC-mediated IL-17 production in memory T cells (Beelen et al., Immunity 2007). Obviously, it has been known for a long time that IL-1β and IL-23 are required for this response. We regret that the reviewer does not understand how 48 h activated DCs can still produce cytokines, but our experiments clearly show that this is the case or otherwise there would not be any TH cell development at all, either in our co-cultures with memory T cells or naive T cells.

The same is true for the naïve T cell culture, the authors write that many more naïve T cells have to be used to set up the culture and to receive sufficient amounts for analysis. In my experience this indicates that the T cells poorly grow and many may eventually die.
The reviewer has misunderstood our comment -we meant that when we analyze our outgrown T cells after 5 days of co-culture (as we did for the naive T cells) instead of allowing an 11-14 days proliferation period (as we did for the memory T cells), we need to start our experiments with more T cells to obtain the same amount of cells for our analyses. So it is not the case that naive T cells grow poorly compared to memory T cells but that the short culture period of 5 days results in less cells for analysis. If required we can also perform the DC-memory T cell co-cultures in 5 days to show that there is no difference between outgrowth of memory or naive T cells.
3. Stainings -In fact the stainings of the naïve T cell cultures also appear untypically to me: in my experience cytokine stainings give rarely such clear round shaped populations (Fig 2a, b) compare also to Fig 2 e, f). Have the same antibodies been used in 2 a,b vs e.f ?. Have doublets been excluded? Here I would like to see the original gates, scatter gates, … We have provided all the original scatter plots and gating strategies for Fig. 2a,b and Fig. 2e,f as pdf attachments and we can include these in the manuscript. We have used the same antibodies throughout the entire manuscript and thus also in Fig. 2a,b vs Fig. 2e,f. Indeed, when analyzing the outgrown T cells after 5 days DC-naive T cell co-cultures, we had to exclude doublets as these cells are still very activated and cluster together. When analyzing the outgrown T cells after 11-14 day DC-memory T cell co-cultures, there are hardly any doublets present as these cells have been cultured until becoming quiescent.

-Especially figure 2 m) appears very untypical to me but maybe I misunderstand it: the dot plots are intracellular staining for IFNg and IL-10 from T cells from one culture? These two populations have then been further analyzed for the genes shown in the histograms?
If this is the case I have severe concerns, whether these data are technically well generated. I never saw IL-10 and IFN-g staining in T cells in two separated populations like this, not to say that this is not possible. This must be an artefact. Also intracellular stainings for transcription factors in our hands never give such homogenous peaks. Also here the authors have to provide the gating, dot plots (double stainings?), etc to make that plausible.
The dot plots are indeed intracellular triple stainings for IFNγ and IL-10 from IL-17+ cells sorted from one culture and these two populations have been further analyzed for the genes shown in the histograms. Importantly, the FACS result is certainly not an artefact, as the two separated populations of IL-10+ and IFNγ+ cells within the IL-17+ cells coincide with the real time PCR analyses we did on sorted IL-17+IFNγ-and IL-17+IFNγ+ cells that likewise show that IL-10 expression is mostly absent in the IL-17+IFNγ+ fraction (Fig. 2k). We see this distinction also when analyzing by real-time PCR the sorted IL-17+IFNγ-and IL-17+IFNγ+ cells after 5 days DCnaive T cell co-cultures (Fig. 2k).
We are surprised that the reviewer doubts our transcription factor stainings. These stainings are done on cells that have been allowed to proliferate until becoming quiescent and have had time to develop the signature profiles to the their full potential. Why would there need to be a broad distribution of transcription factor expression levels between these cells? The remarks of the reviewer indicate that they analyze transcription factor expression much sooner after the start of TH cell development. This could easily explain why they observe more spread in their transcription factor expression patterns. We could even argue that this time effect is visible in the real time PCR analyses of the DC-naive T cell co-cultures vs the DC-memory T cell cocultures: the '5 days old' IL-17+IFNγ+ cells produce significantly less TBX21 (which encodes transcription factor T-bet) and IFNG than the '11-14 days old' IL-17+IFNγ+ cells (Fig. 2k and  Supplementary Fig. 2b,c). Differences in culture conditions will affect TH cell development, and we find it somewhat insulting to simply call this an artefact.
We have added all original scatter plots and gating strategies for Fig. 2m as pdf attachments.

Decision Letter, first revision:
Subject: Decision on Nature Immunology submission NI-A32231A-Z

Message: 22nd Mar 2022
Dear Dr. Geijtenbeek, Thank you for your response to the referees' comments on your article, "Dectin-1 directs non-pathogenic TH17 polarization by regulating release of active TGFβ via tightly controlled type I IFN responses". Although we are interested in the possibility of publishing your study in Nature Immunology, the issues raised by the referees need to be addressed.
Please revise along the lines specified in your letter and per our conversation. At resubmission, please include a "Response to referees" detailing, point-by-point, how you addressed each referee comment. If no action was taken to address a point, you must provide a compelling argument. This response will be sent back to the referees along with the revised manuscript.
Please include a revised version of any required reporting checklist. It will be available to referees to aid in their evaluation. The Reporting Summary can be found here: https://www.nature.com/documents/nr-reporting-summary.pdf When submitting the revised version of your manuscript, please pay close attention to our href="https://www.nature.com/nature-research/editorial-policies/image-integrity">Digital Image Integrity Guidelines.</a> and to the following points below: --that unprocessed scans are clearly labelled and match the gels and western blots presented in figures.
--that control panels for gels and western blots are appropriately described as loading on sample processing controls --all images in the paper are checked for duplication of panels and for splicing of gel lanes.
Finally, please ensure that you retain unprocessed data and metadata files after publication, ideally archiving data in perpetuity, as these may be requested during the peer review and production process or after publication if any issues arise.
Please use the link below to submit your revised manuscript and related files: [REDACTED] <strong>Note:</strong> This URL links to your confidential home page and associated information about manuscripts you may have submitted, or that you are reviewing for us. If you wish to forward this email to co-authors, please delete the link to your homepage.
We hope to receive your revised manuscript within 2-3 months. If you cannot send it within this time, please let us know. We will be happy to consider your revision so long as nothing similar has been accepted for publication at Nature Immunology or published elsewhere.
Please do not hesitate to contact me if you have any questions or would like to discuss these revisions further.
Nature Immunology is committed to improving transparency in authorship. As part of our efforts in this direction, we are now requesting that all authors identified as 'corresponding author' on published papers create and link their Open Researcher and Contributor Identifier (ORCID) with their account on the Manuscript Tracking System (MTS), prior to acceptance. ORCID helps the scientific community achieve unambiguous attribution of all scholarly contributions. You can create and link your ORCID from the home page of the MTS by clicking on 'Modify my Springer Nature account'. For more information please visit please visit <a href="http://www.springernature.com/orcid">www.springernature.com/orcid</a>.
We look forward to seeing the revised manuscript and thank you for the opportunity to review your work. The authors took a serious effort to repond to my comments. In particular the repetition of differentiation experiments with naive T cells (which was also suggested by the two other reviewers) has been performed. The rigor and the flow of the manuscript has much improved by this set of experiments.
I also appreciate the authors' effort to revisit the experimental set-up of Zielinski et al. The data that is is provided in Reviewer Fig. 1-1. is important and should go into the manuscript (at least as supplementary data) since it will indeed help the field to understand these obvious discrepancies in both manuscripts. The authors have addressed most of my points raised in the first review. My biggest concern was about the actual mechanisms underlying the induction of IFNg/IL-17 in human T cells. The addition of data on naïve Th cell differentiation was an important and essential point. To my knowledge the mechanisms of dectin-1 mediated induction of human Th17 cells is also new and could be followed in more detail. However, the data provided so far still do not fully convince me and I have especially some technical concerns regarding intracellular cytokine stainings, which in my eyes prevent publication, if not clarified.
1. General quality of the culture system: In my previous review I raised the point that it is difficult to understand the co-culture system the authors call "T cell outgrowth assay" (14 days, 2 day preactivation of DCs).
The authors now show that DCs stimulated for 3h and 48h have different effects on T cells. This is ok and interesting. However, the authors still do not provide any data about the growth/survival of the T cells throughout the culture. It is well known that during culture small subsets may grow out especially if the overall proliferation is not homogenous. Thus standard parameters should be cell survival, expansion, viability allowing to estimate whether the cultures are comparable. I still disagree with the authors and would say that cytokine induction should be visible early, after a few days. Otherwise it would make biologically no sense. This is important to understand whether we really talk about induction of IFNg/IL-17 or just better survival, e.g. due to more costimulation by dectin1 matured DC. As previously mentioned, I don´t understand why human memory T cells activated for 14 days should not contain any Th17 cells (Fig 2 e, f). Sallusto and colleagues showed nicely that Th17 and Th17*(IFNg) cells and other Th subsets can be directly sorted from blood according to chemokine receptor expression profile, and these cells produce a high amount of signature cytokines. I still think it is required to sort different memory subsets before coculture to distinguish between induction or survival.

Th17 induction
Furthermore I still don´t understand which signals the 48h activated DC mediate to induce IL-17 (or alternatively: support better expansion of pre-existing Th17 cells). The authors write in the response letter that this has been previously been shown to depend on IL-1,-23 but then they should show that DCs after 48h still produce these cytokines and that they are relevant for the Th17 induction. By selectively blocking these cytokines and also IL-12 it can also be excluded that IFNg induction is not driven by IL-12 (as mentioned before to me it looks overall like strong induction of IFNg in all cells not only in the Th17 cells).
The same is true for the naïve T cell culture, the authors write that many more naïve T cells have to be used to set up the culture and to receive sufficient amounts for analysis. In my experience this indicates that the T cells poorly grow and many may eventually die.
3. Stainings -In fact the stainings of the naïve T cell cultures also appear untypically to me: in my experience cytokine stainings give rarely such clear round shaped populations (Fig 2a, b) compare also to Fig 2 e, f). Have the same antibodies been used in 2 a,b vs e.f ?. Have doublets been excluded? Here I would like to see the original gates, scatter gates, … -Especially figure 2 m) appears very untypical to me but maybe I misunderstand it: the dot plots are intracellular staining for IFNg and IL-10 from T cells from one culture? These two populations have then been further analyzed for the genes shown in the histograms? If this is the case I have severe concerns, whether these data are technically well generated. I never saw IL-10 and IFN-g staining in T cells in two separated populations like this, not to say that this is not possible. This must be an artefact. Also intracellular stainings for transcription factors in our hands never give such homogenous peaks. Also here the authors have to provide the gating, dot plots (double stainings?), etc to make that plausible.

Response to Reviewer #1
The authors took a serious effort to repond to my comments. In particular the repetition of differentiation experiments with naive T cells (which was also suggested by the two other reviewers) has been performed. The rigor and the flow of the manuscript has much improved by this set of experiments. Fig. 1-1. is important and should go into the manuscript (at least as supplementary data) since it will indeed help the field to understand these obvious discrepancies in both manuscripts.

I also appreciate the authors' effort to revisit the experimental set-up of Zielinski et al. The data that is is provided in Reviewer
We thank the reviewer for the positive comments. As requested, we have included the data from Reviewer Fig. 1-1 in the manuscript in Supplementary Fig. 9. We thank the reviewer for pointing out these mistakes. We have rectified them in the manuscript.

The Authors have satisfactorily addressed the concerns raised by this reviewer in the first round of revision. Congratulations to the Authors on a nice study!
We thank the reviewer for the positive comments!

Response to Reviewer #3
The authors have addressed most of my points raised in the first review. My biggest concern was about the actual mechanisms underlying the induction of IFNg/IL-17 in human T cells. The addition of data on naïve Th cell differentiation was an important and essential point. To my knowledge the mechanisms of dectin-1 mediated induction of human Th17 cells is also new and could be followed in more detail. However, the data provided so far still do not fully convince me and I have especially some technical concerns regarding intracellular cytokine stainings, which in my eyes prevent publication, if not clarified.

In my previous review I raised the point that it is difficult to understand the co-culture system the authors call "T cell outgrowth assay" (14 days, 2 day preactivation of DCs). The authors now show that DCs stimulated for 3h and 48h have different effects on T cells. This is ok and interesting.
However, the authors still do not provide any data about the growth/survival of the T cells throughout the culture. It is well known that during culture small subsets may grow out especially if the overall proliferation is not homogenous. Thus standard parameters should be cell survival, expansion, viability allowing to estimate whether the cultures are comparable.
As requested, we have now included proliferation and survival data as well as expansion curves for both naive and memory CD4 + T cells that were cocultured with curdlan-or C. albicans-primed DCs in Supplementary Fig. 3. These data show that primed but not immature DCs in the DC-naive T cell cocultures (in the absence of SEB) induced proliferation of a small percentage of T cells, likely in an Agdependent manner. During DC-memory T cell cocultures, which are performed in the presence of SEB, both immature and primed DCs induced proliferation of the majority of the T cells, in an Agindependent manner. Either inhibition or enhancement of type I IFN responses during DC priming, despite changing the cytokine expression profile of the primed DCs, did not alter the observed proliferation and survival profiles of the activated T cells (either naive or memory) during cocultures. Since the cocultures are comparable with regard to proliferation and survival, it seems unlikely that changes in these parameters are responsible for the observed differences in IL-17/IFNγ expression by T cells. We have also clarified this in the manuscript.

I still disagree with the authors and would say that cytokine induction should be visible early, after a few days. Otherwise it would make biologically no sense.
We apologize for the confusion as we previously agreed with the reviewer that cytokine induction in T cells should be visible after a few days: we showed strong cytokine induction in naive T cells after 5 days of coculture with primed DCs (Fig. 2a,b and Supplementary Fig. 9 -numbering corresponding to revised manuscript). We have now also included data after 5 days of DC-memory T cell cocultures, in which we also observed a clear cytokine induction (Supplementary Fig. 5). We have clarified this in the manuscript. This is important to understand whether we really talk about induction of IFNg/IL-17 or just better survival, e.g. due to more costimulation by dectin1 matured DC. As previously mentioned, I don´t understand why human memory T cells activated for 14 days should not contain any Th17 cells (Fig 2 e, f). Sallusto and colleagues showed nicely that Th17 and Th17*(IFNg) cells and other Th subsets can be directly sorted from blood according to chemokine receptor expression profile, and these cells produce a high amount of signature cytokines. I still think it is required to sort different memory subsets before coculture to distinguish between induction or survival.
We apologize for the confusion but we do detect TH17 cells after coculture of memory T cells with immature DCs, although at low levels (<1%) and with donor variation (compare Fig. 2e and 2f). Like Sallusto and colleagues, we have sorted TH17 cells from blood (Fig. 2i,j) and these cells constitute only a small fraction: we isolate ~ 100.000 IL-17 + cells from 15 million CD4 + T cells (this varies per donor), which is <1%. These numbers are also in line with literature (Dagur et al., 2019). After homogenous proliferation due to the presence of SEB, but without specific induction (as observed with our immature DCs), only those <1% will produce IL-17 after re-stimulation, which is exactly what we see (Fig. 2e,f).
We agree with the reviewer that it is important to distinguish whether outgrowth of IL17/IFNγexpressing cells is due to induction or better survival. Since proliferation and survival numbers did not differ between immature and primed DCs (Supplementary Fig. 3), our data suggest that the induction of IL-17 + cells after restimulation of memory T cells cocultured with primed DCs is due to the differentiation of memory T cells into TH17 cells due to dectin-1 cues, and not due to differences in survival. It would indeed be interesting to further examine the plasticity of Th cells and investigate whether a specific memory T cell subset differentiates into TH17 cells but we feel that this is beyond the scope of our manuscript.

Th17 induction
Furthermore I still don´t understand which signals the 48h activated DC mediate to induce IL17 (or alternatively: support better expansion of pre-existing Th17 cells). The authors write in the response letter that this has been previously been shown to depend on IL-1,-23 but then they should show that DCs after 48h still produce these cytokines and that they are relevant for the Th17 induction. By selectively blocking these cytokines and also

it can also be excluded that IFNg induction is not driven by IL-12 (as mentioned before to me it looks overall like strong induction of IFNg in all cells not only in the Th17 cells).
We have determined the expression of IL-1β, IL-23, IL-6 and IL-12 within the supernatant of (un)stimulated DCs over time. We have added these data in Reviewer Fig. 1. While IL-1β and IL-6 expression can be detected after 8 h already, IL-23 and IL-12 expression takes longer. After 24 h, the expression levels of all four cytokines in the supernatant reach a plateau level. Interestingly, after washing the DCs after 48 h, we observe that primed DCs still produce all cytokines. Moreover, when we add either naive or memory T cells, the induction of all cytokines is enhanced. Thus, DCs still produce all cytokines after 48 h and these cytokines will be involved in instructing the T cells. As requested, we have performed blocking experiments and our data show that both IL-1β and IL-23, but not IL-12, produced as a result of dectin-1 signaling, are required for the induction of IL-17 expression in both naive and memory T cells. We have included these data (Supplementary Fig. 5) and clarified our findings in the manuscript.
The same is true for the naïve T cell culture, the authors write that many more naïve T cells have to be used to set up the culture and to receive sufficient amounts for analysis. In my experience this indicates that the T cells poorly grow and many may eventually die.
We are afraid that the reviewer has misunderstood our comment -we meant that when we analyze our outgrown T cells after 5 days of co-culture (as we did for the naive T cells) instead of allowing an 11-14 days proliferation period (as we did for the memory T cells), we need to start our experiments with more T cells to obtain the same amount of cells for our analyses. The expansion curves in Supplementary Fig. 3c and g showcase the differences in cell numbers we obtained. The proliferation data in Supplementary Fig. 3 show that both our naive and memory T cell cocultures are growing nicely.

Stainings -
In fact the stainings of the naïve T cell cultures also appear untypically to me: in my experience cytokine stainings give rarely such clear round shaped populations (Fig 2a, b) compare also to Fig 2 e, f). Have the same antibodies been used in 2 a,b vs e.f ?. Have doublets been excluded? Here I would like to see the original gates, scatter gates, … We thank the reviewer for this question. We have now enlisted the help of Dr. Ester Remmerswaal within our department, who is an expert in both T lymphocytes and flow cytometry analyses, to ensure proper analyses of our data.
Previously, we did not exclude doublets or debris from our memory T cell analyses and this has caused an overestimation of double positive IL-17 + IFNγ + cells in our experiments. We have added both our previous and new analyses, including gating strategies and scatter plots for Fig. 2e and f in Reviewer Fig. 2 (added as a separate pdf file), to illustrate the differences. Interestingly, undisturbed dectin-1 signaling leads to even less pathogenic TH17 cell induction. Disturbances in type I IFN responses still lead to a significant increase in IL-17 + IFNγ + T cells. Thus, strictly regulated dectin-1 signaling seems even more important to prevent the generation of pathogenic TH17 cells. We have now included our gating strategy in the manuscript in Supplementary Fig. 4. This does not affect our analyses of sorted TH17 cells after DC-memory T cell experiments (real-time PCR experiments, FACS stainings) as here the correct gating strategies had been applied (see also Reviewer Fig. 2 (added as a separate pdf file)).
We have used the same antibodies throughout the entire manuscript and thus also in Fig. 2a,b  vs Fig. 2e,f. We have no explanation for the different shape of the cytokine stainings and can only speculate that this is an intrinsic characteristic of the naive T cells. Also we observe differences in the shape of the cytokine stainings between the resting and blast cells after re-stimulation (Supplementary Fig. 4). We have also re-analyzed our DCnaive T cell coculture data as previously we excluded blast cells from our naive T cell analyses, erroneously identifying these as doublets. We have added both our previous and new analyses, including gating strategies and scatter plots for Fig. 2a and b in Reviewer Fig. 2 (added as a separate pdf file), to illustrate the differences. As the IL-17 and IFNγ expression levels of the blast cells differ from that of the resting cells after re-stimulation, it became necessary to separate both populations before our analyses of the IL-17 + IFNγand IL-17 + IFNγ + cells, otherwise IL-17 + IFNγblast cells would overlap with IL-17 + IFNγ + resting cells. As the blast cell population varies per experiment (10-40% of the lymphocyte single cell gate), this effect has smaller or larger consequences depending on the experiment. We have now included our gating strategy in the manuscript in Supplementary Fig. 4.

-
Especially figure 2 m) appears very untypical to me but maybe I misunderstand it: the dot plots are intracellular staining for IFNg and IL-10 from T cells from one culture? These two populations have then been further analyzed for the genes shown in the histograms? If this is the case I have severe concerns, whether these data are technically well generated. I never saw IL-10 and IFN-g staining in T cells in two separated populations like this, not to say that this is not possible. This must be an artefact. Also intracellular stainings for transcription factors in our hands never give such homogenous peaks. Also here the authors have to provide the gating, dot plots (double stainings?), etc to make that plausible.
The dot plots are indeed intracellular triple stainings for IFNγ and IL-10 from IL-17 + cells sorted from one culture and these two populations have been further analyzed for the genes shown in the histograms. Importantly, the FACS results are certainly not an artefact, as the two separated populations of IL-10 + and IFNγ + cells within the IL-17 + cells coincide with the real time PCR analyses we did on sorted IL-17 + IFNγand IL-17 + IFNγ + cells that likewise show that IL-10 expression is mostly absent in the IL-17 + IFNγ + fraction (Fig. 2k). We see this distinction also when analyzing mRNA expression of the sorted IL-17 + IFNγand IL-17 + IFNγ + cells after 5 days DC-naive T cell co-cultures by real-time PCR (Fig. 2k).
We have included all original scatter plots and gating strategies for Fig. 2m in Reviewer Fig. 2 (added as a separate pdf file). We can only speculate on the differences between our stainings and those of the reviewer. Possibly, the more homogenous expression of transcription factors is due to the fact that these T cells have been grown until they become quiescent and as such have developed the signature profiles to the their full potential. It is possible that the reviewer analyses transcription factor expression much sooner after the start of TH cell development, which would perhaps lead to more spread in their transcription factor expression patterns as the signature is not yet fully developed.
Reviewer Fig. 1 | IL-1β, IL-23, IL-6 and IL-12 expression over time in supernatants of primed DCs. ELISA for quantification of IL-1β, IL-6, IL-23 and IL-12 in the supernatant of DCs at the indicated times after stimulation with curdlan or C. albicans CBS2712. After 48 h, either naive (Tn) T cells were added to the medium or DCs was washed and resuspended in fresh medium, either without or with naive or memory (Tm) T cells. Data represent mean ± s.d. of 3 independent donors.
Thanks to the authors for providing all the additional data and controls and for answering all my questions. This was very helpful and improved the manuscript.
However, there are still two last points related to figure 2. The data in figure 2 provide such a black and white picture of pathogenic versus non pathogenic Th17 cells (which is a concept very much discussed whether these phenotypes exist in vivo) that I have difficulties to fully comprehend this. But this is more based on experience rather than on specific points which look critical, therefore I would like to state that as my personal opinion.
We thank the reviewer for the positive comments on the new data in our revised manuscript.

Fig 2 i, j If I understand correctly: Here the authors analyse IL-17+ cells sorted ex vivo from PMA/iono stimulated T cells from healthy donors and CD patients, sorted for IL-17 and subsequently analyzed for the various genes by RT-PCR? The authors find an almost black and white difference between IL-17+ cells from healthy donors and CD patients (n=4) with respect to genes expressed by so called pathogenic and non-pathogenic Th17 cells.
The analysis judged from the gating strategies looks fine, but such a marked difference is surprising and I am not aware that something similarly drastic has previously been described. My impression from the overall literature and personal experience is that only mild differences between patients and healthy can be observed. So the authors should at least comment on this striking observation in their assay system (which is sophisticated) and discrepancy to published data (or prove me wrong with examples from the literature). They should also discuss that additional functional data (e.g. cytokine secretion data, i.c. stainings, more and defined patients …) may be necessary to support this finding in the future.
The reviewer is correct that we have analysed IL-17+ cells sorted from healthy donors and acute untreated CD patients. We have also sorted IL-17+ cells from CD patients that are in remission, and the differences with healthy donors are much milder (Reviewer Figure 1), suggesting that the striking differences between healthy and acute CD patients might be because the CD patients used in the manuscript are untreated and acute. We are also not aware of any study that has performed a similar technical challenging analysis, which makes it very interesting and we will further pursue this in another study.
As requested, we can discuss the observed differences between healthy and CD patients in the manuscript and also discuss that further study is necessary to investigate the striking Th17 phenotype in acute CD patients. The high frequencies of IL-10 population and clear separation might be due to our DC stimulation and T cell outgrowth assay. As our data show curdlan and C. albicans induce a very strong Th17 phenotype. The uniform staining of IL-10 might be caused by the procedure as the cells are permeabilized/fixed, sorted and permeabilized/fixed again (using a special kit for transcription factor staining) before staining. However, the IL-10 expression data are supported by the mRNA expression profiles (Fig. 2I). We can discuss this in the manuscript.

Decision Letter, second revision:
Subject: Decision on Nature Immunology submission NI-A32231B Message: Dear Dr. Geijtenbeek, Thank you for your response to the reviewers' comments on your manuscript "Dectin-1 directs non-pathogenic TH17 polarization by regulating release of active TGFβ via tightly controlled type I IFN responses". We are happy to inform you that if you revise your manuscript appropriately in response to the referees' comments and our editorial requirements your manuscript should be publishable in Nature Immunology.
Please revise your manuscript to address the reviewer's comments and as outlined in your letter. At resubmission, please include a point-by-point response to the referees' comments, noting the pages and lines where the changes can be found in the revision. Please highlight the changes in the revised manuscript as well. Please note that articles for Nature Immunology have a word limit of 4000 words for the introduction, results and discussion and a limit of 50 references for the main text. The discussion should not exceed 800 words.
We are trying to improve the quality and transparency of methods and statistics reporting in our papers (please see our editorial in the May 2013 issue). Please update the Life Sciences Reporting Summary, and supplements if applicable, with any information relevant to any new experiments and upload it (as a Related Manuscript File) along with the files for your revision. If nothing in the checklist has changed, please upload the current version again.
TRANSPARENT PEER REVIEW Nature Immunology offers a transparent peer review option for new original research manuscripts submitted from 1st December 2019. We encourage increased transparency in peer review by publishing the reviewer comments, author rebuttal letters and editorial decision letters if the authors agree. Such peer review material is made available as a supplementary peer review file. Please state in the cover letter 'I wish to participate in transparent peer review' if you want to opt in, or 'I do not wish to participate in transparent peer review' if you don't. Failure to state your preference will result in delays in accepting your manuscript for publication.
Please note: we allow redactions to authors' rebuttal and reviewer comments in the interest of confidentiality. If you are concerned about the release of confidential data, please let us know specifically what information you would like to have removed. Please note that we cannot incorporate redactions for any other reasons. Reviewer names will be published in the peer review files if the reviewer signed the comments to authors, or if reviewers explicitly agree to release their name. For more information, please refer to our <a href="https://www.nature.com/documents/nr-transparent-peer-review.pdf" target="new">FAQ page</a>. ORCID Nature Immunology is committed to improving transparency in authorship. As part of our efforts in this direction, we are now requesting that all authors identified as 'corresponding author' on published papers create and link their Open Researcher and Contributor Identifier (ORCID) with their account on the Manuscript Tracking System (MTS), prior to acceptance. ORCID helps the scientific community achieve unambiguous attribution of all scholarly contributions. For more information please visit <a href="http://www.springernature.com/orcid">www.springernature.com/orcid</a>.
Before resubmitting the final version of the manuscript, if you are listed as a corresponding author on the manuscript, please follow the steps below to link your account on our MTS with your ORCID. If you don't have an ORCID yet, you will be able to create one in minutes. If you are not listed as a corresponding author, please ensure that the corresponding author(s) comply.
follow these instructions. Non-corresponding authors do not have to link their ORCIDs, but please note that it will not be possible to add/modify ORCIDs at proof. Thus, if they wish to have their ORCID added to the paper, they must also follow the above procedure prior to acceptance.
To support ORCID's aims, we only allow a single ORCID identifier to be attached to one account. If you have any issues attaching an ORCID identifier to your Manuscript Tracking System account, please contact the <a href="http://platformsupport.nature.com/">Platform Support Helpdesk</a>.
We hope that you will support this initiative and supply the required information. Should you have any query or comments, please do not hesitate to contact our editorial assistant at immunology@us.nature.com. Nature Immunology has now transitioned to a unified Rights Collection system which will allow our Author Services team to quickly and easily collect the rights and permissions required to publish your work. Once your paper is accepted, you will receive an email in approximately 10 business days providing you with a link to complete the grant of rights. If you choose to publish Open Access, our Author Services team will also be in touch at that time regarding any additional information that may be required to arrange payment for your article.
Please note that you will not receive your proofs until the publishing agreement has been received through our system.
For information regarding our different publishing models please see our <a href="https://www.springernature.com/gp/open-research/transformative-journals"> Transformative Journals </a> page. If you have any questions about costs, Open Access requirements, or our legal forms, please contact ASJournals@springernature.com..
In recognition of the time and expertise our reviewers provide to Nature Immunology's editorial process, we would like to formally acknowledge their contribution to the external peer review of your manuscript entitled "Dectin-1 directs non-pathogenic TH17 polarization by regulating release of active TGFβ via tightly controlled type I IFN responses". For those reviewers who give their assent, we will be publishing their names alongside the published article.
When you are ready to submit your revised manuscript, please use the URL below to submit the revised version: [REDACTED] We hope to receive your revised manuscript in 10 days, by 5th Aug 2022. Please let us know if circumstances will delay submission beyond this time. If you have any questions please do not hesitate to contact me. However, there are still two last points related to figure 2. The data in figure 2 provide such a black and white picture of pathogenic versus non pathogenic Th17 cells (which is a concept very much discussed whether these phenotypes exist in vivo) that I have difficulties to fully comprehend this. But this is more based on experience rather than on specific points which look critical, therefore I would like to state that as my personal opinion.

Fig 2 i, j
If I understand correctly: Here the authors analyse IL-17+ cells sorted ex vivo from PMA/iono stimulated T cells from healthy donors and CD patients, sorted for IL-17 and subsequently analyzed for the various genes by RT-PCR? The authors find an almost black and white difference between IL-17+ cells from healthy donors and CD patients (n=4) with respect to genes expressed by so called pathogenic and non-pathogenic Th17 cells. The analysis judged from the gating strategies looks fine, but such a marked difference is surprising and I am not aware that something similarly drastic has previously been described. My impression from the overall literature and personal experience is that only mild differences between patients and healthy can be observed. So the authors should at least comment on this striking observation in their assay system (which is sophisticated) and discrepancy to published data (or prove me wrong with examples from the literature). They should also discuss that additional functional data (e.g. cytokine secretion data, i.c. stainings, more and defined patients …) may be necessary to support this finding in the future.
Fig 2m: These are stainings for IL-10 and IFNg within T cells sorted for IL-17 + after culture (according to the raw data files provided). From the dot plots and gating strategies, I did not identify any obvious issues. However, from all my experience of 25 years cytokine staining of human and murine T cells focusing on IL-10 from Th1/Th17 subsets, I have never observed such well separated IFNg versus IL-10 producing populations of Th17 cells and such extremely high frequencies of 70% IL-10 (uniform staining). All other cytokine stainings in the manuscript look different, although there are no other IL-10 stainings provided in the manuscript.

Response to Reviewer #3
Thanks to the authors for providing all the additional data and controls and for answering all my questions. This was very helpful and improved the manuscript.
However, there are still two last points related to figure 2. The data in figure 2 provide such a black and white picture of pathogenic versus non pathogenic Th17 cells (which is a concept very much discussed whether these phenotypes exist in vivo) that I have difficulties to fully comprehend this. But this is more based on experience rather than on specific points which look critical, therefore I would like to state that as my personal opinion.
We thank the reviewer for the positive comments on the new data in our revised manuscript. The authors find an almost black and white difference between IL-17+ cells from healthy donors and CD patients (n=4) with respect to genes expressed by so called pathogenic and non-pathogenic Th17 cells. The analysis judged from the gating strategies looks fine, but such a marked difference is surprising and I am not aware that something similarly drastic has previously been described.
My impression from the overall literature and personal experience is that only mild differences between patients and healthy can be observed. So the authors should at least comment on this striking observation in their assay system (which is sophisticated) and discrepancy to published data (or prove me wrong with examples from the literature). They should also discuss that additional functional data (e.g. cytokine secretion data, i.c. stainings, more and defined patients …) may be necessary to support this finding in the future.
The reviewer is correct that we have analyzed IL-17+ cells sorted from healthy donors and CD patients. These CD patients had acute and untreated immunopathogenesis. We have also sorted IL-17+ cells from CD patients that are in remission, and the differences with healthy donors are much less pronounced, suggesting that the striking differences between healthy and CD donors as shown in the manuscript are because of the disease status of the donors. We are also not aware of any study that has performed a similar technical challenging analysis, which makes it very interesting and we will further pursue this in another study. We have now added the data from the treated CD patients that show only mild differences with healthy persons in Supplementary Fig. 6a and described this observation in the text, page 8, line 184-186. However, from all my experience of 25 years cytokine staining of human and murine T cells focusing on IL-10 from Th1/Th17 subsets, I have never observed such well separated IFNg versus IL-10 producing populations of Th17 cells and such extremely high frequencies of 70% IL-10 (uniform staining). All other cytokine stainings in the manuscript look different, although there are no other IL-10 stainings provided in the manuscript.
The high frequencies of IL-10-expressing cells and clear separation might be due to our DC stimulation and T cell outgrowth assay: our data show that curdlan and C. albicans induce a very strong TH17 phenotype, while the setup of the outgrowth assay (especially the prolonged culture) is beneficial for obtaining high frequencies of induced subsets. The 1 uniform staining of IL-10 might be caused by the procedure as the cells are permeabilized/fixed, sorted and permeabilized/fixed again (using a special kit for transcription factor staining) before staining. However, the IL-10 expression data are supported by the mRNA expression profiles (Fig. 2l). Thank you for your patience as we've prepared the guidelines for final submission of your Nature Immunology manuscript, "Dectin-1 directs non-pathogenic TH17 polarization by regulating release of active TGFβ via tightly controlled type I IFN responses" (NI-A32231C). Please carefully follow the step-by-step instructions provided in the attached file, and add a response in each row of the table to indicate the changes that you have made. Please also check and comment on any additional marked-up edits we have proposed within the text. Ensuring that each point is addressed will help to ensure that your revised manuscript can be swiftly handed over to our production team.

Decision
The handling editor would like to start working on your revised paper, with all of the requested files and forms, as soon as possible and has requested that you return them by September 22nd. Please get in contact with us if you anticipate delays.
When you upload your final materials, please include a point-by-point response to any remaining reviewer comments and please make sure to upload your checklist.
If you have not done so already, please alert us to any related manuscripts from your group that are under consideration or in press at other journals, or are being written up for submission to other journals (see: https://www.nature.com/nature-portfolio/editorialpolicies/plagiarism#policy-on-duplicate-publication for details).
In recognition of the time and expertise our reviewers provide to Nature Immunology's editorial process, we would like to formally acknowledge their contribution to the external peer review of your manuscript entitled "Dectin-1 directs non-pathogenic TH17 polarization by regulating release of active TGFβ via tightly controlled type I IFN responses". For those reviewers who give their assent, we will be publishing their names alongside the published article.
Nature Immunology offers a Transparent Peer Review option for new original research manuscripts submitted after December 1st, 2019. As part of this initiative, we encourage our authors to support increased transparency into the peer review process by agreeing to have the reviewer comments, author rebuttal letters, and editorial decision letters published as a Supplementary item. When you submit your final files please clearly state in your cover letter whether or not you would like to participate in this initiative. Please note that failure to state your preference will result in delays in accepting your manuscript for publication.

Cover suggestions
As you prepare your final files we encourage you to consider whether you have any images or illustrations that may be appropriate for use on the cover of Nature Immunology.
Covers should be both aesthetically appealing and scientifically relevant, and should be supplied at the best quality available. Due to the prominence of these images, we do not generally select images featuring faces, children, text, graphs, schematic drawings, or collages on our covers.
We accept TIFF, JPEG, PNG or PSD file formats (a layered PSD file would be ideal), and the image should be at least 300ppi resolution (preferably 600-1200 ppi), in CMYK colour mode.
If your image is selected, we may also use it on the journal website as a banner image, and may need to make artistic alterations to fit our journal style.
Please submit your suggestions, clearly labeled, along with your final files. We'll be in touch if more information is needed. Nature Immunology has now transitioned to a unified Rights Collection system which will allow our Author Services team to quickly and easily collect the rights and permissions required to publish your work. Approximately 10 days after your paper is formally accepted, you will receive an email in providing you with a link to complete the grant of rights. If your paper is eligible for Open Access, our Author Services team will also be in touch regarding any additional information that may be required to arrange payment for your article.
You will not receive your proofs until the publishing agreement has been received through our system. Please note that <i>Nature Immunology</i> is a Transformative Journal (TJ). Authors may publish their research with us through the traditional subscription access route or make their paper immediately open access through payment of an article-processing charge (APC). Authors will not be required to make a final decision about access to their article until it has been accepted. <a href="https://www.springernature.com/gp/openresearch/transformative-journals">Find out more about Transformative Journals</a>. If you have any questions about costs, Open Access requirements, or our legal forms, please contact ASJournals@springernature.com.
Authors may need to take specific actions to achieve <a href="https://www.springernature.com/gp/open-research/funding/policycompliance-faqs"> compliance</a> with funder and institutional open access mandates. If your research is supported by a funder that requires immediate open access (e.g. according to <a href="https://www.springernature.com/gp/open-research/plan-scompliance">Plan S principles</a>) then you should select the gold OA route, and we will direct you to the compliant route where possible. For authors selecting the subscription publication route, the journal's standard licensing terms will need to be accepted, including <a href="https://www.springernature.com/gp/open-research/policies/journalpolicies">self-archiving policies</a>. Those licensing terms will supersede any other terms that the author or any third party may assert apply to any version of the manuscript.
Please use the following link for uploading these materials: [REDACTED] If you have any further questions, please feel free to contact me. I am delighted to accept your manuscript entitled "Dectin-1 directs non-pathogenic TH17 polarization by regulating release of active TGFβ via tightly controlled type I IFN responses" for publication in an upcoming issue of Nature Immunology.
Over the next few weeks, your paper will be copyedited to ensure that it conforms to Nature Immunology style. Once your paper is typeset, you will receive an email with a link to choose the appropriate publishing options for your paper and our Author Services team will be in touch regarding any additional information that may be required.
After the grant of rights is completed, you will receive a link to your electronic proof via email with a request to make any corrections within 48 hours. If, when you receive your proof, you cannot meet this deadline, please inform us at rjsproduction@springernature.com immediately.
You will not receive your proofs until the publishing agreement has been received through our system.
Due to the importance of these deadlines, we ask that you please let us know now whether you will be difficult to contact over the next month. If this is the case, we ask you provide us with the contact information (email, phone and fax) of someone who will be able to check the proofs on your behalf, and who will be available to address any lastminute problems.
Acceptance is conditional on the data in the manuscript not being published elsewhere, or announced in the print or electronic media, until the embargo/publication date. These restrictions are not intended to deter you from presenting your data at academic meetings and conferences, but any enquiries from the media about papers not yet scheduled for publication should be referred to us.
Please note that <i>Nature Immunology</i> is a Transformative Journal (TJ). Authors may publish their research with us through the traditional subscription access route or make their paper immediately open access through payment of an article-processing charge (APC). Authors will not be required to make a final decision about access to their article until it has been accepted. <a href="https://www.springernature.com/gp/openresearch/transformative-journals">Find out more about Transformative Journals</a>.
Authors may need to take specific actions to achieve <a href="https://www.springernature.com/gp/open-research/funding/policycompliance-faqs"> compliance</a> with funder and institutional open access mandates. If your research is supported by a funder that requires immediate open access (e.g. according to <a href="https://www.springernature.com/gp/open-research/plan-scompliance">Plan S principles</a>) then you should select the gold OA route, and we will direct you to the compliant route where possible. For authors selecting the subscription publication route, the journal's standard licensing terms will need to be accepted, including <a href="https://www.springernature.com/gp/open-research/policies/journalpolicies">self-archiving policies</a>. Those licensing terms will supersede any other terms that the author or any third party may assert apply to any version of the manuscript.
If you have any questions about our publishing options, costs, Open Access requirements, or our legal forms, please contact ASJournals@springernature.com Your paper will be published online soon after we receive your corrections and will appear in print in the next available issue. Content is published online weekly on Mondays and Thursdays, and the embargo is set at 16:00 London time (GMT)/11:00 am US Eastern time (EST) on the day of publication. Now is the time to inform your Public Relations or Press Office about your paper, as they might be interested in promoting its publication. This will allow them time to prepare an accurate and satisfactory press release. Include your manuscript tracking number (NI-A32231D) and the name of the journal, which they will need when they contact our office.
About one week before your paper is published online, we shall be distributing a press release to news organizations worldwide, which may very well include details of your work. We are happy for your institution or funding agency to prepare its own press release, but it must mention the embargo date and Nature Immunology. Our Press Office will contact you closer to the time of publication, but if you or your Press Office have any enquiries in the meantime, please contact press@nature.com.
Also, if you have any spectacular or outstanding figures or graphics associated with your manuscript -though not necessarily included with your submission -we'd be delighted to consider them as candidates for our cover. Simply send an electronic version (accompanied by a hard copy) to us with a possible cover caption enclosed.
To assist our authors in disseminating their research to the broader community, our SharedIt initiative provides you with a unique shareable link that will allow anyone (with or without a subscription) to read the published article. Recipients of the link with a subscription will also be able to download and print the PDF.
As soon as your article is published, you will receive an automated email with your shareable link.
You can now use a single sign-on for all your accounts, view the status of all your manuscript submissions and reviews, access usage statistics for your published articles and download a record of your refereeing activity for the Nature journals.
If you have not already done so, we strongly recommend that you upload the step-by-step protocols used in this manuscript to the Protocol Exchange. Protocol Exchange is an open online resource that allows researchers to share their detailed experimental know-how. All uploaded protocols are made freely available, assigned DOIs for ease of citation and fully searchable through nature.com. Protocols can be linked to any publications in which they are used and will be linked to from your article. You can also establish a dedicated page to collect all your lab Protocols. By uploading your Protocols to Protocol Exchange, you are enabling researchers to more readily reproduce or adapt the methodology you use, as well as increasing the visibility of your protocols and papers. Upload your Protocols at www.nature.com/protocolexchange/. Further information can be found at www.nature.com/protocolexchange/about . Please note that we encourage the authors to self-archive their manuscript (the accepted version before copy editing) in their institutional repository, and in their funders' archives, six months after publication. Nature Portfolio recognizes the efforts of funding bodies to increase access of the research they fund, and strongly encourages authors to participate in such efforts. For information about our editorial policy, including license agreement and author copyright, please visit www.nature.com/ni/about/ed_policies/index.html An online order form for reprints of your paper is available at <a href="https://www.nature.com/reprints/authorreprints.html">https://www.nature.com/reprints/author-reprints.html</a>. Please let your coauthors and your institutions' public affairs office know that they are also welcome to order reprints by this method.