TGF-β signaling in Th17 cells promotes IL-22 production and colitis-associated colon cancer

IL-22 has dual functions during tumorigenesis. Short term IL-22 production protects against genotoxic stress, whereas uncontrolled IL-22 activity promotes tumor growth; therefore, tight regulation of IL-22 is essential. TGF-β1 promotes the differentiation of Th17 cells, which are known to be a major source of IL-22, but the effect of TGF-β signaling on the production of IL-22 in CD4+ T cells is controversial. Here we show an increased presence of IL-17+IL-22+ cells and TGF-β1 in colorectal cancer compared to normal adjacent tissue, whereas the frequency of IL-22 single producing cells is not changed. Accordingly, TGF-β signaling in CD4+ T cells (specifically Th17 cells) promotes the emergence of IL-22-producing Th17 cells and thereby tumorigenesis in mice. IL-22 single producing T cells, however, are not dependent on TGF-β signaling. We show that TGF-β, via AhR induction, and PI3K signaling promotes IL-22 production in Th17 cells.


Reviewer #2, expertise in IL-22 (Remarks to the Author):
The role of TGF-beta in the production of IL-22 in CD4(+) T cells still remains controversial. To clarify this important issue both scientifically and clinically, this manuscript employs a series of reporter mouse systems. The reporter mouse systems include triple reporter mice for detection of IL-17A, IL-22, and Foxp3 and fate-map double reporter mice for identifying IL-22-producing cells without versus with prior IL-17A expression. The authors initially confirm the increase of TGF-beta expression and IL-17A(+) IL-22(+) T cells in the tissue of human colorectal cancer. In vitro experiments using mouse T cells reveal an ability of TGF to promote the emergence of IL-17A(+) IL-22(+) CD4(+) T cells. In vivo experiments using T cells in which TGF-beta signal is impaired due to the overexpression of dominant negative TGF-betaRII as well as T cells in which TGFbetaR2 is specifically deleted in Th17 cells (under control of IL-17A promoter) demonstrate that TGF-beta induces the differentiation of IL-17(+)  and IL-17(+)IL-22(+), but not IL-17(-)IL-22(+), T cells. The contribution of IL-17(+)IL-22(+) T cells for promoting carcinogenesis pathway are also shown by transferring T cells with intact versus impaired TGF-beta signaling into immune-deficient colitis-associated cancer model without or with endogenous IL-22. A series of in vitro experiments propose that, in addition to TGF-beta signaling, Ahr activation and strong TCR signaling medicated by PI3K are required for the differentiation of IL-17(+)IL-22(+) T cells. Based on these data, the authors conclude that TGF-beta signaling in Th17 cells promotes IL-22 production and colitis associated cancer. Overall, this manuscript is carefully designed with a substantial depth to minimize potential problems and provide a novel insight into Th17 biology.
Here are some specific comments: The authors emphasize colitis-associated cancer throughout the manuscript. However, it is unclear whether colitis-associated cancer or sporadic cancer was used for the analysis of human samples. It becomes increasingly apparent that CD4(+) T cells expressing both IL-17 and Foxp3 develop particularly in the intestine (Immunity 2019, p212; Nat Immunol 2019, p471). Since the authors used a triple reporter mouse system capable of detecting not only IL-17A and IL-22 but also Foxp3, it may be appreciated if they show whether IL-17(+) IL-22(+) T cells express Foxp3 or not.
Reviewer #3, expertise in T helper cell differentiation (Remarks to the Author): The authors of this manuscript aimed to resolve some of the controversy surrounding the role of TGFβ mediated regulation of IL-22 production in CD4 T cells. Using transgenic reporter mice for IL17A and IL22 they clearly demonstrate in vitro that TGF-β and strong TCR stimulation coupled with AhR ligands, promotes the emergence of IL-17+IL-22+ T cells as well as the production of IL-22 in already differentiated Th17 cells. They relate these results to in vivo models of intestinal tumorigenesis demonstrating impaired TGF-β signaling in T cells reduces IL-22 production in Th17 cells and subsequent tumor burden. Finally, they show both IL-17A+IL-22+ producing T cells as well as TGF-β levels are increased in human CRC samples compared to normal adjacent tissue suggesting the conclusions drawn from their in vitro and mouse models are also likely true in the context of human disease. However, the manuscript lacks the evidence showing a causal effect of IL-17+IL-22+ cells and the development of colorectal cancer and the roles of IL-17+IL-22-or IL-17-IL-22+ in tumorigenesis are unclear.
For the AOM/DSS CRC mouse model, the authors transferred congenically marked wild type or TGF-β-DNR transgenic (Tg) CD4+ T cells into Rag1-/-mice. This is concerning because of the lymphopenic niche in Rag1-/-mice will lead to homeostatic proliferation of the transferred cells. It is important to determine if the same results would be achieved if the cells were transferred into mice with fully intact adaptive immune systems.
In Figure 5 in addition to using Ahr inhibitor, the data need to be substantiated by using AhR-deficient CD4+ T cells.
Other comments: From the methods, MACS kits were used for cell isolations but no post-enrichment purity was shown. In general, FACS data on IL-22 expression in transfer models need improvement. It needs special attention for Citrobacter rodentium model in which IL-22 can barely be detected. 1

Comments by the editor
We hope you will find the referees' comments useful as you decide how to proceed. Should further experimental data or analysis allow you to address these criticisms within six months, we would be happy to look at a substantially revised manuscript.
However, please bear in mind that we would not consider the manuscript for publication in the absence of major revisions.
We are grateful for the overall positive assessment of our manuscript and for the opportunity to submit a revised version. The reviewer's comments have helped to further improve our study and we provide a point by point reply of our revisions below.
Specifically, the revisions must include (but are not limited to): 1. Addressing all technical concerns of our referees, including statistical analyses and variability of IL-22+ IL-17+ cell frequency across experiments; please also provide absolute numbers.
As requested, we have included the statistical analysis and absolute cell numbers.
As for the variability of IL-17A+IL-22+ T cells, we agree with the reviewer that we observed fewer IL-17A and IL-22 producing cells in the experiment shown in Figure 4 compared to the experiment shown in Figure 3. This effect was most prominent for the IL-17A+ IL-22+ double positive cells, but the IL-17 and IL-22 single producing cell frequencies were also lower.
We would, however, like to point out that this was not due to a technical problem because the isolation (we had similar cell numbers and similar amounts of living cells), staining, and analysis were similar between both sets of experiments.
Of note, the experiments shown in Figure 3 and Figure 4 were not performed as a head to head comparison, but over the course of five years. Indeed, the experiments using the TGFβ-DNR transgenic mice were performed in 2015, while the experiments using the IL-17A Cre x TGFBR2 Flox/Flox within the last year. During this time our mice, including the Rag1-/-mice, were moved to another breeding facility and the diet was also changed. Of course, this also had a major impact on the intestinal microbiota. Indeed, we are currently analyzing the role of different intestinal microbiota compositions on the emergence of IL-17+IL-22+ producing cells and have found a major role of SFB on the emergence of IL-22 producing cells (independent of the known effects on IL-17A production). However, we believe that the study of these environmental effects goes beyond the scope of this manuscript.
However, since we were aware of the potentially high impact of the intestinal microbiota, we intentionally used littermate controls and co-transfer systems for the mouse in vivo experiments. This allowed us not only to discriminate between cell extrinsic and cell intrinsic effects, but also to control for the intestinal microbiota.
In light of this, we now discuss this in the revised version of the manuscript: 'Indeed, we did observe a high variability in the frequency of IL-17+IL-22+ CD4+ T cells over the course of the in vivo experiments. This effect might be due to different intestinal microbiota compositions of the mouse lines used. We therefore used littermate controls and co-transfer experiments in order to control for microbial effects. ' (page: 13, line: 453) 2. Causative evidence that TGF-β signaling in Th17 cells promotes tumorigenesis by inducing IL-22; or more generally, functional evidence that the T-cell source of IL-22 impacts physiology or pathology. We realize that the second point requires longterm in vivo experiments with complex genetics, and that their results may not support the hypothesis that IL-22 plays a distinct and critical role in IL-17producers. If this turns out to be the case, we can consult with our colleagues at Communication Biology on whether the degree of advance would be sufficient for them to offer publication.
We do agree that this is a key point, and as a result, we have indeed performed several additional experiments which support the note that 'TGF-β signaling in Th17 cells promotes tumorigenesis by inducing IL-22'.
First, we found that TGF-β signaling in CD4+ T cells is important for the emergence of IL-22 producing Th17 cells (Figure 1 and 3). Thus, impaired TGF-beta signaling led to a reduction of IL-22 producing Th17 cells and correlated with a decreased tumor load in the colon ( Figure   3). Of note, IL-22 single producing T cells were not affected by the loss of TGF-β signaling.
Second, we found that TGF-β signaling, specifically in Th17 cells promotes IL-22 production in vitro ( Figure 5) and in vivo ( Figure 4). Accordingly, mice with a Th17 cell specific blockade of TGF-beta signaling showed reduced tumorigenesis in the colon ( Figure 4).
Third, in order to finally prove that TGF-β signaling promotes tumorigenesis by inducing IL-22 producing Th17 cells, we repeated the experiment shown in Figure 3 in an IL-22 free environment. To this end, we transferred Il22-/-and TGF-β-DNR transgenic x Il22-/-CD4+ T cell into Rag1-/-x Il22-/-mice. As control, we transferred wild type and TGF-β-DNR transgenic CD4+ T cells. Indeed, we could confirm that mice receiving wild type T cells showed a higher tumor load compared to mice receiving TGF-β-DNR transgenic CD4+ T cells (new Figure 3, below). This effect cannot be due to a difference in IL-22 single producing cells, as we found similar frequencies of these cells in both groups ( Figure 3A). Of note, in an IL-22 free environment mice receiving Il22-/-or TGF-β-DNR transgenic x Il22-/-CD4+ T cell showed an equal tumor load (new Figure 3, below) indicating that the observed effect is indeed IL-22 dependent.
Taken together, these experiments show that TGF-β signaling in CD4+ T cells and specifically, Th17 cells is in fact critical for the emergence of IL-22 producing Th17 cells and for the promotion of colorectal cancer in an IL-22 dependent manner. . Lines indicate mean +/-sem; Tukey´s multiple comparisons test was performed (P<0.05) to assess the significance. Source data are provided as a Source data file.

TGF-β in differentiated Th17 cells that promotes IL-22 production and CAC development is of great interest.
We thank this reviewer for the very positive assessment of our work and for recognizing the novelty of our study. We agree with this reviewer and -as suggested-clarified this speculation:

Similarly, the percentage of regulatory T cells (Tregs) was decreased in normal
colon tissue but not in tumors of CAC model (Fig S4). While this may not be the main point of this study, one can benefit from clearer description of the data in the text section.
We agree and we modified the text accordingly: 'As expected [24], the frequency of Foxp3+ CD4+ T cells ( Figure S4) was also reduced in TGF-β-DNR transgenic CD4+ T cells compared to wild type control in normal colon. However, this was not the case in the tumor tissue.' (page: 7, line: 236). We agree that this is a key point and we have thus performed further experiments to clarify this aspect. Indeed, we now provide several lines of evidence supporting the note that TGF-β signaling in Th17 cells promotes tumorigenesis by inducing IL-22.

The authors showed that when TGF-β signaling was blocked in T cells (
First, we found that TGF-β signaling in CD4+ T cells is important for the emergence of IL-22 producing Th17 cells (Figure 1 and 3). Thus, impaired TGF-beta signaling led to a reduction of IL-22 producing Th17 cells and correlated with a decreased tumor load in the colon ( Figure   3). Of note, IL-22 single producing T cells were not affected by the loss of TGF-β signaling.
Second, we found that TGF-β signaling specifically in Th17 cells promotes IL-22 production in vitro ( Figure 5) and in vivo ( Figure 4). Accordingly, mice with Th17 cell specific blockade of TGF-beta signaling showed reduced tumorigenesis in the colon ( Figure 4).
Third, in order to finally prove that TGF-β signaling promotes tumorigenesis by inducing IL-22 producing Th17 cells, we repeated the experiment shown in Figure  . Lines indicate mean +/-sem; Tukey´s multiple comparisons test was performed (P<0.05) to assess the significance. Source data are provided as a Source data file. Figure 5A, the levels of IL-10 appear similar between groups of weak v.s. strong

TCR stimulation. Is the difference significant?
We agree that the difference in IL-10 levels is small. However, it is still significant (p< 0.0001). We have now included a statistical analysis in Figure 5A.

The authors claimed that TGF-β induced the expression of AhR and thus IL-22 production in Th17 cells. The data showed that AhR is induced upon stimulation of naïve T cells with both IL-6 and TGF-β. Is TGF-β alone effective in AhR induction?
Could the authors observe increased AhR expression when differentiated Th17 cells were stimulated with TGF-β (as shown in Figure 5C)?
We did not study the effect of TGF-β alone on the induction of AhR by naïve T cells, since this in the presence of mAb CD3 (3 µg/ml), mAb CD28 (0.5 µg/ml), APCs and either TGF-β alone, or blocking TGF-β antibody or TGF-β + IL-6 as positive control. After three days, we measured AhR mRNA expression. Our results indicate that the expression of AhR in Th17 cells is maintained by the addition of TGF-β to the culture but is not further increased compared to AhR expression in differentiated Th17 cells before the re-culture. Interestingly, AhR expression decreased significantly in the presence of the TGF-β blocking antibody. These data suggest that the TGF-β, which is present in the culture is sufficient and necessary to maintain AhR expression in differentiated Th17 cells. Finally, the addition of TGF-β and IL-6 to already differentiated Th17 cells did not further increase AhR expression (Reply Figure 1).
We would be happy to include these data in the manuscript upon request.

In the text, the authors hypothesized that TGF-β may promote IL-22 production via
the induction of PI3K pathway together with TCR engagement. However, Figure 6 only showed the role of strong TCR stimulation in the activation of PI3K/Ca2+

pathway. Is TGF-β signal still required for PI3K activation of Ca2+ influx?
TGF-β signal is not required for PI3K activation of Ca2+ influx. Indeed, as shown in Figure 6 A, B, C, activation of PI3K and Ca 2+ influx was seen in the absence of TGF-β. Of note in the performed experiments, we used a calibration buffer to resuspend the cells, which is serum free and therefore also TGF-β free. Thus, TGF-β signaling is not essential for PI3K activation and Ca2+ influx. -

Since the T cells were stimulated with strong TCR engagement (which favors IL-22 production in these cells), why do they still require TGF-β to activate the same pathway?
Our data indicate that TGF-β signaling is important for Ahr induction, which then is essential to induce IL-22 production ( Figure 5).
We have modified the text to clarify this point:

IL-17(+)IL-22(+), but not IL-17(-)IL-22(+), T cells. The contribution of IL-17(+)IL-22(+) T cells for promoting carcinogenesis pathway are also shown by transferring T cells with intact versus impaired TGF-beta signaling into immune-deficient colitis-associated cancer model without or with endogenous IL-22. A series of in vitro experiments
propose that, in addition to TGF-beta signaling, Ahr activation and strong TCR signaling medicated by PI3K are required for the differentiation of IL-17(+)IL-22(+) T

cells. Based on these data, the authors conclude that TGF-beta signaling in Th17 cells promotes IL-22 production and colitis associated cancer. Overall, this manuscript is carefully designed with a substantial depth to minimize potential problems and provide a novel insight into Th17 biology.
We thank this reviewer for carefully reading our manuscript and for his very positive assessment of our work.
Specific comments: 1. The authors emphasize colitis-associated cancer throughout the manuscript.
However, it is unclear whether colitis-associated cancer or sporadic cancer was used for the analysis of human samples.
We clarified this point as requested: 'In order to corroborate this finding, we measured the concentration of total and active TGF-β1 in tissue lysates from colon tumor and normal adjacent tissue from a cohort of patients with sporadic CRC (Table S1).' (page:5, line: 168) Regarding the variability of IL-17A+IL-22+ T cells, the reviewer is right that we observed fewer IL-17A and IL-22 producing cells in the experiment shown in Figure 4 compared to the experiment shown in Figure 3. This effect was most prominent for the IL-17A+ IL-22+ double positive cells, but the IL-17 and IL-22 single producing cell frequencies were also lower.

The average of IL-17A(+)IL-22(+) in the control (WT/WT) shown in
We would, however, like to point out that this was not due to a technical problem because the isolation (we had similar cell numbers and similar amounts of living cells), staining, and analysis were similar between both sets of experiments.
In line with this point made by the reviewer, we also repeated the experiment shown in Figure   4 (which is based on the co-transfer of CD4+ T cells from IL-17A Cre x TGFBR2 Flox/Flox or IL-17A Cre x TGFBR2 Wt/Wt littermate control mice into Rag1-/-mice) using IL-17A Cre x TGFBR2 Flox/Flox and IL-17A Cre x TGFBR2 Wt/Wt littermate control mice directly and thus avoiding the transfer (Reply Figure 2). This time, the frequencies of IL-17 producing and IL-22 producing cells are comparable to those shown in Figure 4 (which is also based on the use of these mice) and again lower than the ones we observed in the experiments shown in Figure  3, in which we used the TGF-β-DNR transgenic mice and littermate controls. Thus, these lower frequencies are not due to the transfer into a lymphopenic host.
Of note, the experiments shown in Figure 3, Figure 4, and Reply Figure 2 were not performed as a head to head comparison, but over the course of five years. Indeed, the experiments using the TGF-β-DNR transgenic mice were performed in 2015, while the experiments using the IL-17A Cre x TGFBR2 Flox/Flox within the last year. During this time our mice, including the Rag1-/-mice, were moved to another breeding facility and the diet was also changed. Of course, this also had a major impact on the intestinal microbiota. Indeed, we are currently analyzing the role of different intestinal microbiota compositions on the emergence of IL-17+IL-22+ producing cells, and have found a major role of SFB on the emergence of IL-22 producing cells (independent of the known effects on IL-17A production). However, we believe that the study of these environmental effects goes beyond the scope of this manuscript.
However, since we were aware of the potentially high impact of the intestinal microbiota, we intentionally used littermate controls and co-transfer systems for the mouse in vivo experiments. This allowed us not only to discriminate between cell extrinsic and cell intrinsic effects, but also to control for the intestinal microbiota between WT and transgenic cells.
In light of this, we now discuss this in the revised version of the manuscript: Colitis associated colon cancer was induced in IL-17A Cre x TGFBR2 fl//fl or IL-17A Cre x TGFBR2 wt/wt mice. Production of IL-17A and IL-22 by T cells from colon was analyzed by flow cytometry. Each dot represents one mouse (TGFBR2 Wt/Wt n=9; TGFBR2 Flox/Flox n=7). Lines indicate mean +/-sem; Mann-Whitney test was performed to assess significance.

It becomes increasingly apparent that CD4(+) T cells expressing both IL-17 and
Foxp3 develop particularly in the intestine (Immunity 2019, p212; Nat Immunol 2019, p471). Since the authors used a triple reporter mouse system capable of detecting not only IL-17A and IL-22 but also Foxp3, it may be appreciated if they show whether

IL-17(+) IL-22(+) T cells express Foxp3 or not.
We agree and we have performed the requested analysis. These data are depicted in Reply Figure 3. We could observe that the presence of IL-17A+Foxp3+ T cells in the tumors was not affected by the impaired TGF-b signaling. Moreover, IL-17A+IL-22+Foxp3+ T cells were almost not detectable in the tumors. We would be happy to include these data in the manuscript upon request. We thank this reviewer for carefully reading our manuscript.

17-IL-22+ in tumorigenesis are unclear.
We agree that this is a key point and we have thus performed further experiments to clarify this question. Indeed, we now provide several lines of evidence supporting the note that TGFβ signaling in Th17 cells promotes tumorigenesis by inducing IL-22.
First, we found that TGF-β signaling in CD4+ T cells is important for the emergence of IL-22 producing Th17 cells (Figure 1 and 3). Thus, impaired TGF-beta signaling led to a reduction of IL-22 producing Th17 cells and correlated with a decreased tumor load in the colon ( Figure   3). Of note, IL-22 single producing T cells were not affected by the loss of TGF-β signaling.
Second, we found that TGF-β signaling specifically in Th17 cells promotes IL-22 production in vitro ( Figure 5) and in vivo (Figure 4). Accordingly, mice with Th17 cell specific blockade of TGF-beta signaling showed reduced tumorigenesis in the colon (Figure 4).
Third, in order to finally prove that TGF-β signaling promotes tumorigenesis by inducing IL-22 producing Th17 cells, we repeated the experiment shown in Figure    Lines indicate mean +/-sem; Tukey´s multiple comparisons test was performed (P<0.05) to assess the significance. Source data are provided as a Source data file.

For the AOM/DSS CRC mouse model, the authors transferred congenically marked
wild type or TGF-β-DNR transgenic (Tg) CD4+ T cells into Rag1-/-mice. This is concerning because of the lymphopenic niche in Rag1-/-mice will lead to homeostatic proliferation of the transferred cells. It is important to determine if the same results would be achieved if the cells were transferred into mice with fully intact adaptive immune systems.
We thank the reviewer for this comment. Indeed, we confirmed the data using IL-17A cre x TGFBRII fl/fl mice (Figure 4 B + C and Reply Figure 2, below) avoiding the transfer.
The reviewer is right that the lymphopenic niche in Rag1-/-mice will lead to homeostatic proliferation of the transferred cells. Nevertheless, we intentionally used this approach since the TGF-β-DNR transgenic mice have impaired TGF-β signaling, not only on CD4+ T cells but also on CD8+ T cells, which are also important in tumor immunity. Unfortunately, we cannot do the transfer directly into immune competent mice: TGF-β-DNR transgenic mice were generated using the human CD2 promoter. Thus, these cells will be rejected if transferred into immune competent mice. We actually tried this experiment, and we could not monitor the cells for more than two weeks after transfer. A) Naïve T cells were differentiated in the presence of anti-CD3 (3μg/ml), APCs and indicated factors. Relative Ahr, Rorc, cmaf, Il22, Il17a, Il10 mRNA expression on day 2 of the culture was measured using RT-PCR. 2-way ANOVA, Tukey's multiple comparisons test. B) Naïve T cells from Foxp3 mRFP x IL-17A eGFP x IL-22 sgBFP reporter mice were cultured under Th17 polarizing condition (mAb IL-4 (10 µg/ml), mAb INF-γ (10 µg/ml), mAb CD3 (3 µg/ml), mAb CD28 (0.5 µg/ml), IL-6 (10 ng/ml), TGF-β1 (1 ng/ml) and FICZ (100mM)) with increasing amounts of AhR antagonist (as indicated) for 4 days.  Other comments: 1. From the methods, MACS kits were used for cell isolations but no post-enrichment purity was shown.
We did use MACS isolation kits in order to enrich for naive T cells. Overall the purity was about 85% as shown in the representative Reply Figure 4 bellow. We have included this information in the revised version of the manuscript: 'To that end, we differentiated naïve T cells, purified with an efficiency around 85% using MACS beads, from wild type mice under different conditions, that have been previously reported to modulate IL-22 production in vitro' (page: 5, line: 187)

Reply Fig. 4: Enrichment of CD4+ naïve T cells using Magnetic cell separation (MACS) kits.
Mouse splenocytes were depleted from CD25 and CD44 and followed by positive CD4 selection. Frequency of enriched naïve CD4 + T cells is shown.

In general, FACS data on IL-22 expression in transfer models need improvement. It needs special attention for Citrobacter rodentium model in which IL-22 can barely be detected.
We agree with the reviewer and we improved the FACS data shown in the manuscript including the one from the Citrobacter rodentium model ( Figure S5). The revised manuscript by Perez et al reported a novel mechanism by which TGF-β promotes IL-22 production from Th17 cells and therefore augments the development of colitis associated colorectal cancer. In additional to the work described in their first submission, the authors now also show that: 1) TGF-β promotes the production of IL-22 in Th17 cells; 2) IL-22 from Th17 cells are important for colonic tumor development; and 3) TGF-β signaling to differentiated Th17 cells is required for the maintenance of AhR expression. These additional data were backed by compelling in vivo and in vitro evidence, and supports the overarching conclusion of the paper. The authors have addressed all questions of this reviewer, and the work is now in a great shape for publication.
Reviewer #2 (Remarks to the Author): In the initial submission, three concerns were raised by this reviewer regarding the type of cancer (colitis-associated versus sporadic), inconsistent date from figure to figure, and potential expression of Foxp3 in IL-17(+) IL-22(+) T cells. These concerns have been satisfactorily addressed in this revised manuscript by additional data.
Reviewer #3 (Remarks to the Author): The authors have eloquently addressed all reviewer comments; therefore, I believe this manuscript is worthy of acceptance to Nature Communications.
The causal the roles of IL-17+IL-22+ vs IL-17-IL-22+ in the development of CRC have been clarified by performing further experiments which provide evidence to support their claims. By transferring WT vs Il22-/-and TGF-β-DNR transgenic x Il22-/-CD4+ T cell into an Il22-/-environment the authors demonstrate more tumor load in the mice that received WT but similar tumor load (less) in all other groups suggesting the effect is indeed IL-22 dependent. Additionally, the authors accept the limitation of their study concerning the contribution of IL-17A and have revised the manuscript accordingly.
As requested, by including an Ahr knockout control, the authors confirmed their results showing Ahr signaling mediated the effects of TGF-b1 on the development of IL-17+IL-22+ T cells.
Minor comments were also addressed well.
Post-enrichment purity is now shown and the FACS data showing IL-22 expression was improved.