Cell type-specific enhancers regulate IL-22 expression in innate and adaptive type 3 lymphoid cells

Domain: biology
Published: 2026-03-14
Editorial outcome: minor_revision

Abstract
IL-22, a signature cytokine for type 3 lymphoid cells, including T helper 17/22 (Th17/22) and type 3 innate lymphoid cells (ILC3), mediates epithelial homeostasis and protective pathogen responses in barrier tissues. Upon dysregulation, IL-22 can drive chronic inflammatory diseases, yet little is known about transcriptional elements modulating its expression. Here, we identify two enhancers, E22-1 and E22-2, with distinct capacities for regulating Il22 expression in type 3 lymphoid cells. Both enhancers are necessary for protection from Citrobacter rodentium infection and for the onset of IL-22-mediated psoriasis. E22-2 is specifically required for IL-22 expression in ILC3s, while E22-1 functions in both Th17/22 and ILC3. The ILC3 specificity of E22-2 is attributed to the presence of multiple Runx3 sites and the lack of a functional RORγt motif. We conclude that Th17/22 and ILC3 cells use distinct cis-elements to differentially regulate IL-22 expression, while orchestrating homeostatic protection and pathogen defense in barrier tissues.

Introduction
The cytokine IL-22 functions as an important regulator of epithelial homeostasis and antimicrobial responses in barrier tissues, including the gastrointestinal tract, lung, liver, skin, and breast1–5. The primary sources of IL-22 are type 3 innate lymphoid cells (ILC3) and their adaptive counterparts, Th17/22 cells, which are collectively referred to as type 3 immune cells since they have similar effector programs1,4,6–9. In addition, IL-22 is produced by other innate cells, such as NKT17 and -T cells in response to danger signals associated with infection or inflammation. The cognate receptor for IL-22 is a heterodimer of IL-22R1 and IL-10R2, which is expressed on epithelial and stromal cells, as well as other cells of non-hematopoietic origin10. Binding of IL-22 to its receptor primarily triggers the JAK1-STAT3 signaling pathway, which induces expression of anti-microbial peptides (AMP), chemokines, anti-apoptotic molecules, and genes involved in cell proliferation, tissue repair, and regeneration11. As such, IL-22 is pivotal in the initiation of antimicrobial responses, resolution of inflammation, and restoration of barrier integrity.

In this regard, IL-22 and its sources play a multi-faceted role in maintaining intestinal health and defense against pathogens that infect gut tissues, processes that have been especially well-studied in mice infected by Citrobacter rodentium8,12–15. This enteric bacterium attaches to mucosal surfaces in the colon, disrupting barrier function, and it serves as a pathogenesis model for E. coli-induced colitis in humans. Indeed, C. rodentium infection in IL-22 deficient mice leads to severe colitis and high mortality rates12. Recent studies have shown that IL-22 derived from innate lymphoid cells, such as ILC3s, plays a crucial role in controlling the early stages of C. rodentium infection, while antigen-specific CD4+ Th17/22 populations expand and become important sources of IL-22 during later stages of infection, aiding in final clearance of the pathogen13,14.

Conversely, perturbations in IL-22 levels have been associated with several cancers, as well as inflammatory and autoimmune disorders, emphasizing the pathogenic role of IL-2216,17. In patients suffering from psoriasis, a chronic skin inflammatory disorder, enhanced levels of IL-22 in skin and blood have been correlated with disease severity18. Likewise, in mice, imiquimod induces a psoriasis-like skin inflammation that is driven by IL-22 and IL-1719–21. However, the relevant contribution of innate and adaptive immune cell types producing these pathogenic type 3 cytokines remains unresolved. In this regard, many studies have underscored the potential of IL-22 as a therapeutic target for chronic inflammatory conditions involving barrier tissues and ongoing clinical trials are focused on drugs that either impact the production, neutralization, or downstream signaling of IL-2222.

While the effects of IL-22 on epithelial cells have been well-studied, significant gaps remain in our understanding about cis-regulatory circuits that control homeostatic IL-22 expression or its induction in lymphoid cells during normal or pathogenic responses. At least some ILC3s express IL-22 at a steady state in healthy tissues, whereas CD4+ T helper cells normally require TCR and/or cytokine activation for its expression. IL-23 is the major cytokine that induces IL-22 in both innate and adaptive immune cells, which, in conjunction with IL-1, leads to optimal IL-22 production12,17,23,24. These pathways activate STAT3 and the signature type 3 transcription factor, RORγt, which are known to augment the expression of Il22 at the RNA level. However, understanding how cis-regulatory circuits coordinate disparate signals to tune Il22 gene expression in different type 3 immune cells inhabiting normal and pathogenic microenvironments remains a key goal, especially given the therapeutic potential of regulating Il22 expression.

To address this knowledge gap, we here perform a CRISPRi screen of potential cis-regulatory elements embedded within the 1.5 Mb Mdm1-Il22-Ifng locus using an ILC3 cell model. We identify two enhancers conserved between humans and mice, E22-1 and E22-2, which differentially augment Il22 expression levels in innate versus adaptive lymphoid cells. Together, these enhancers are required for homeostatic, protective, and pathogenic IL-22 expression in vivo. Remarkably, E22-2 is highly specific for ILC3s and a small percentage of -T cells. This activity profile distinguishes E22-2 from other type 3 lymphoid enhancers, which normally function in both Th and ILC counterparts. Mechanistically, the underlying architecture of E22-2 restricts its activity to ILC3s, likely to support its role as a homeostatic enhancer of IL-22 expression in barrier tissues. The distinct activities of E22-1 and E22-2 regulate IL-22 expression to balance steady state fitness of barrier tissues, rapid innate immune responses by ILC3s and γδ-T cells, and subsequent antigen-specific responses to pathogens by Th17/22 cells.

Results
CRISPRi screen identifies potential cis-elements for Il22 expression in type 3 lymphoid cells
In both humans and mice, IL22 is situated in a locus that also contains genes encoding the type 1 cytokine IFN-γ, IL-26 (only in humans), and the cell cycle regulator MDM1 (Fig. 1a). These multigenic loci are characterized by a complex regulatory space with many conserved DNA elements that have active chromatin characteristics in lymphoid cells, including a large ILC3-specific super-enhancer (SE) region in both species (Fig. 1b)7. To determine which of these accessible chromatin regions are necessary for regulating Il22 expression in type 3 cells, we performed a CRISPRi screen in the mouse ILC3 cell model, Mnk325. Like primary ILC3s, Mnk3 cells produce high levels of IL-22 and IL-17F in response to the type 3 agonists IL-1 and IL-23. Moreover, accessible chromatin patterns in Mnk3, as observed in ATAC-seq data, largely parallel those reported previously for primary mouse ILC3s (Fig. 1b)26,27.

To perform CRISPRi screens, we engineered a Mnk3 subclone to express a catalytically inactive Cas9 associated with a KRAB chromatin repressor domain (dCas9-KRAB) under tetracycline control, as well as an mCherry reporter (Mnk3i cell line, Fig. 1c). The Mnk3i line was infected with a custom lentiviral sgRNA library (MOI < 0.3) that tiles all accessible chromatin regions in a ~1.5 Mb space surrounding the Il22 locus (Fig. 1b). The accessible regions chosen for this library were identified using ATAC-seq data from a collection of different lymphoid cell types, including Mnk3, the ILC1 model Mnk1, primary type 1 cells (NK, ILC1, and Th1), and primary type 3 cells (ILC3 and Th17)26,27. The lentiviral library also had a number of control sgRNAs that targeted the Il22, Il23r, and Il1rap promoters (positive controls), as well as ATAC-negative regions on chromosome 10 and sgRNAs with no targets in the mouse genome (negative controls). Lentiviral-infected cells were selected with puromycin, followed by treatment with doxycycline (dox) (24 hours), before adding the type 3 agonists IL-1 and IL-23. After 16 additional hours, IL-17F expressing cells were flow sorted into populations that expressed high, medium, low, or no detectable IL-22 (Supplementary Fig. 1a). sgRNA sequences were amplified by PCR from each fraction, which were deep sequenced and analyzed by the MAGeCK pipeline28. The fraction expressing the highest levels of IL-22 yielded low cell numbers and was excluded from further analysis.

Because this was an inhibitory screen, we focused on regions in which sgRNAs were enriched significantly in the IL-22 negative fraction but were depleted in cells expressing IL-22, when compared with sgRNA representation in their counterpart fractions that were not treated with dox (i.e., lacking dCas9-KRAB). The top 10 regions exhibiting these characteristics are shown in Fig. 1d. As expected, sgRNAs targeting promoters of Il22, Il23r, and Il1rap, a component of IL-1 receptor complex, were in this top 10 list, whereas sgRNAs targeting ATAC-negative regions were neither significantly enriched nor depleted in any cell population (Fig. 1d, e and Supplementary Fig. 1b-d). Among the top 10 hits, only two regions showed the expected enrichment-depletion profiles in all three sorted cell fractions, as well as high sequence conservation in mammals. We named these accessible chromatin regions, E22-1, positioned ~32 kb upstream, and E22-2, located ~17 kb upstream of the Il22 promoter (Fig. 1e). Of note, no accessible regions within the ILC3-specific SE were identified using this CRISPRi approach. The lack of hits in the SE could reflect functional redundancy within its individual regulatory elements, or that the SE may be irrelevant for IL-22 expression in this cell model. Notwithstanding, the CRISPRi screen identified E22-1 and E22-2 as candidate cis-elements that activate Il22 expression in type 3 immune cells.

E22-1 and E22-2 function as Il22 enhancers in Mnk3 cells
To further explore whether E22-1 and E22-2 serve as Il22 regulatory elements, we surveyed chromatin profiles in several relevant lymphoid populations (Mnk3, NK, ILC1, ILC3, and Th17 cells), using new (this study) or published ATAC- and H3K27ac-seq data26. The latter histone modification represents an excellent metric for enhancer activity. As shown in Fig. 2a, the distal element, E22-1, was highly accessible in type 3 (ILC3, Th17, and Mnk3) but not in type 1 cells (NK and ILC1), and stimulation of Mnk3 with IL-1+IL-23 further enhanced its H3K27ac. By contrast, E22-2 was accessible only in ILC3s, but not in Th17 or type 1 cells. Like E22-1, H3K27ac at E22-2 was enhanced in Mnk3 upon IL-1+IL-23 treatment. These chromatin data suggest that both E22-1 and E22-2 are enhancers that can be induced by type 3 agonists.

To test for enhancer function, we performed luciferase reporter assays on cloned E22-1 and E22-2 sequences in Mnk3 cells using vectors driven by the mouse Il22 promoter. Neither element augmented expression from the Il22 promoter in unstimulated Mnk3 cells, but treatment with IL-1+IL-23 induced robust enhancer activity for both E22-1 (~4 -fold) and E22-2 (~3-fold) (Fig. 2b). Consistent with chromatin profiling, only E22-1 displayed enhancer activity in the mouse T-cell line, EL4 (Fig. 2c). As expected, neither element enhanced reporter activity in the mouse pre-B cell line, 63-12 (Supplementary Fig. 2a). To further establish their enhancer functions at the endogenous Il22 locus, we introduced sgRNAs that were designed to target either E22-1 or E22-2 in Mnk3i. Induction of dCas9-KRAB mediated significant repression of IL-22 with either enhancer sgRNA compared with a scrambled sgRNA in stimulated Mnk3 cells (Fig. 2d), but none of the sgRNAs affected levels of the other type 3 cytokine, IL-17F (Fig. 2e), whose gene is located on a different chromosome (IL-17A is not expressed in the parental Mnk3 line). In addition, expression of the neighboring gene, Mdm1, was unaffected by CRISPRi-mediated repression of E22-1 or E22-2, whereas Il22 transcripts were significantly diminished (Supplementary Fig. 2b, c).

In some mouse strains, including C57BL/6, 129 and FVB, the region between Il22 and Ifng harbors a pseudogene ~80 kb downstream of Il22, called Iltifb, which is an inverted duplication of Il2229. The Iltifb region also contains a duplicate of E22-2, which we coined twin-E22-2, but has no duplicate of E22-1 (Supplementary Fig. 2d). Our CRISPRi screen did not identify twin-E22-2 as a putative candidate regulating Il22. Nevertheless, the similarities between E22-2 and twin-E22-2 merited direct verification to rule out any role for the latter in Il22 gene regulation. For this purpose, we designed sgRNAs that specifically target either E22-2 or twin-E22-2 and verified their specificities using in vitro cutting assays with Cas9 ribonucleoprotein complexes (Supplementary Fig. 2e). IL-22 expression was unchanged in a Mnk3i line that stably expressed the twin-E22-2 sgRNA (Supplementary Fig. 2f). In contrast, Mnk3i cells expressing the highly specific E22-2 sgRNA exhibited a significant reduction in IL-22 levels. As expected, none of the sgRNAs impacted IL-17F expression (Supplementary Fig. 2g). Collectively, our molecular assays in Mnk3 cells implicate E22-1 and E22-2 as the two most important cis-regulatory elements for Il22 expression in this type 3 lymphoid cell model.

In vivo regulation of Il22 by E22-1 and E22-2
To investigate their functions in vivo, we used CRISPR-Cas9 technology to generate mice lacking E22-1(E22-1), E22-2 (E22-2), or lacking both elements (E22-1/2). The E22-1 and E22-2 mice were created by deletion of 3.5 and 2.5 kb, respectively, spanning regions covered by H3K27ac in Mnk3 cells. The E22-1/2 strain was generated by deleting E22-1 from E22-2 mice using the same sgRNAs. All deletions were confirmed by DNA sequencing and the double knockout was verified to be on the same allele using long-range PCR.

To verify that E22-1 and E22-2 were primary IL-22 enhancers for type 3 cells in vivo, rather than only having importance in the Mnk3 cell model, we first focused on mice lacking both elements. ILC3s were analyzed from small intestine lamina propria (SI-LP), as they are scarce in other tissues. This site contains all three known subsets, namely, NKp46+, CCR6+ (lymphoid tissue inducer cells, LTi), and double negative (DN)30. We observed no significant changes in the frequency of ILC3 subsets in the E22-1/2 mice compared to WT controls (Supplementary Fig. 3a). Bulk SI-LP cells were cultured in the presence or absence of IL-1 and IL-23 for 3.5 h and IL-22 levels were quantified in each ILC3 subset using flow cytometry. As shown in Fig. 3a-c, mice lacking both elements had a ~3-fold reduction in average IL-22 expression, as well as a significant reduction in the percentages of IL-22 positive cells for all three ILC3 subsets (Supplementary Fig. 3b-d). Since IL-22 production by Th cells is minimal at steady state, we examined the effect of deleting the two dominant enhancers by culturing splenic naïve CD4+ T cells under Th22-polarizing conditions for 3 days. As shown in Fig. 3d, there was a dramatic reduction in both levels of IL-22 expression (~4-fold) and percentages of IL-22 positive cells (~7-fold; Supplementary Fig. 3f) for Th22 cells derived from E22-1/2 mice compared with WT animals. Importantly, IL-17A expression remained unaffected (Supplementary Fig. 3e). The Il22 locus is flanked by Ifng, the signature cytokine of Th1 subset. A previous study has shown that these two genes reside in adjacent but separate topologically associated domains, insulated by a CTCF-binding site31. Consistent with their independent regulation, IFN-γ expression in ex vivo polarized Th1 cells was not significantly different in E22-1/2 vs WT mice (Supplementary Fig. 3j).

In addition to ILC3 and Th17/22 cells, IL-22 is produced by NKT17 and -T cells, two other innate immune subsets1,4. To determine the impact of the E22-1/2 mutation on the former subset, we isolated peripheral lymph nodes cells, where they are abundant, and subjected them to overnight stimulation with anti-CD3 and anti-CD28 along with IL-1 and IL-23. Cultured cells were then analyzed by flow cytometry to establish IL-22 expression levels in NKT17 cells. As shown in Fig. 3e and Supplementary Fig. 3g, h, NKT17 cells from E22-1/2 mice had more than 10-fold reduction in IL-22, while IL-17A expression was unaffected. Lastly, -T cells from SI-LP isolates were analyzed after stimulation with IL-1+IL-23. The E22-1/2 cells had ~4-fold reduction in IL-22 compared with -T cells from WT controls (Fig. 3f and Supplementary Fig. 3i).

To test whether the observed reductions in IL-22 expression by type 3 cells was physiologically relevant, we examined the impact of the dual enhancer deletion on IL-22 dependent homeostatic and defense programs in the gut. With regards to the former, IL-22 induces steady-state expression of AMPs by intestinal epithelial cells. As such, we performed RT-qPCR analysis for transcripts corresponding to several AMPs using RNA isolated from terminal ileum of WT and E22-1/2 mice. We observed significantly reduced steady-state levels of transcripts corresponding to IL-22 inducible AMP genes, Reg3, Reg3, Reg3, and Saa1/2, in E22-1/2 mice (Fig. 3g). Consistent with this, we observed a similar reduction in the expression of Reg3, Reg3, and Saa1/2 in the colon of E22-1/2 mice at steady state (Supplementary Fig. 3k), while expression of Reg3α and the gene encoding IL-22 binding protein (Il22bp) were nearly undetectable. To test the functional importance of the E22-1 and E22-2 enhancers in mucosal defense, we infected WT and E22-1/2 mice with C. rodentium. Over the course of 20 days following infection, E22-1/2 mice experienced more pronounced weight loss at early (days 2-4) and late time points (days 10-14) as compared to WT controls (Fig. 3h). The weight loss was also accompanied by a significantly higher fecal bacterial burden on days 12 and 16 post-infection (Fig. 3i). Thus, the E22-1 and E22-2 enhancers are required for attaining levels of IL-22 expression by type 3 immune cells that mediate both steady-state homeostatic protection in the gut and defense against a mucosal pathogen, C. rodentium.

E22-1 and E22-2 both contribute to cell type-specific patterns of IL-22 expression and protection from imiquimod-induced psoriasis
To investigate the individual roles of E22-1 and E22-2, we evaluated IL-22 production by type 3 immune cells in single knockout mice. We did not observe any significant change in the frequency of SI-LP ILC3 subsets in either of the tested mutant mice (Supplementary Fig. 4a and l). Deletion of E22-1 impaired expression of IL-22 in all three subsets of ILC3s, a reduction comparable to that observed in E22-1/2 derived ILC3s (Fig. 4a and Supplementary Fig. 4b-f). We also observed a significant decrease in the levels of IL-22 and the percentage of IL-22 positive Th22 cells after polarization of splenic naïve CD4+ T cells from E22-1 mice, while IL-17A levels remained unchanged (Fig. 4b and Supplementary Fig. 4g, h). In addition, both NKT17 and -T cells from E22-1 mice expressed reduced levels of IL-22 as compared to WT controls (Fig. 4c, d and Supplementary Fig. 4i-k). Overall, the E22-1 mutation phenocopied the double knockout with regards to IL-22 expression in all tested type 3 lymphoid cell subsets.

We next examined the E22-2 single knockout and observed a decrease in IL-22 expression for ILC3 subsets that was comparable to the E22-1 and E22-1/2 mice (Fig. 4e and Supplementary Fig. 4m-q). Importantly, however, IL-22 production was unaffected in Th22 and NKT17 cells from the E22-2 animals (Fig. 4f, 4h, Supplementary Fig. 4r, s, u, and v). As shown in Fig. 4g and Supplementary Fig. 4t, the E22-2 deletion led to a modest, but significant decrease (~2-fold) in IL-22 expression by -T cells. The reduced levels of IL-22 in either E22-1 or E22-2 mice led to a downregulation of Reg3, Reg3, Reg3, and Saa1/2 in intestinal epithelium (Fig. 4i, j). Taken together, E22-1 and E22-2 both contribute to optimal IL-22 production in ILC3s and -T cells; whereas, E22-2 is dispensable for normal induction of this cytokine in Th22 and NKT17 cells, suggesting an ILC3 and -T cell-specific function for E22-2. Surprisingly, deletion of both elements had no additional impact on IL-22 expression in any of the type 3 lymphoid subsets tested, suggesting that the two enhancers are partially compensatory rather than synergistic in ILC3s and -T cells (see discussion).

In addition to its role as a protective cytokine, IL-22 has a pathologic function in inflammatory diseases, such as IBD and psoriasis. These effects are attributable, in part, to its function in inducing pro-inflammatory genes, as well as anti-apoptotic and proliferative pathways. In this regard, IL-22 contributes to the development of psoriasis-like lesions following application of the topical pro-inflammatory agent, imiquimod19. Imiquimod-induced psoriasis is characterized by redness and scaling of the skin, thickening of the epidermal layer from hyperplasia and keratin retention, as well as thickening of the dermis from inflammatory infiltrates and edema. The importance of IL-22 in the pathogenesis of human psoriasis is underscored by a linkage between deficits in IL-22 binding protein, elevated levels of IL-22 in skin, and enhanced incidence of lesions18,32.

To test whether Il22 enhancers with distinct activity profiles differentially affect IL-22 mediated psoriasis, we treated each of the mutant mice with topical administration of imiquimod to their ears. WT mice developed imiquimod-induced psoriasis-like lesions on their ears over a period of 9 days, and this pathology was reduced in each of the enhancer-deleted animals (Supplementary Fig. 5). As a more quantifiable measurement, we found that all mutant mice had reduced thickening of the pinna compared to WT animals (Fig. 5a). These differences were evident when using a modified psoriasis area and severity index (PASI) score, which qualitatively quantifies redness, scaling, and thickness of psoriasis lesions (Fig. 5b-e)33. Imiquimod-treated WT mice also showed a significant increase in the thickness of the epidermal layer compared to E22-1, E22-2, and E22-1/2 mice, when examined histologically (Fig. 5f). Furthermore, there was a significant decrease in Il22 transcript levels in the ear skin of all mutant mice on day 5 of imiquimod-induced psoriasis, as measured by RT-qPCR, whereas Il17a and Il17f transcripts remained comparable to those in WT animals (Fig. 5g-i). Notably, mice lacking the innate cell-specific enhancer, E22-2, had a reduced pathology that was comparable to animals lacking the E22-1 enhancer, which is also functional in Th17/22 cells. These data suggest that IL-22 expressed by ILC3 and -T cells in the skin is sufficient to drive psoriasis pathogenesis in this mouse model.

Cell type-specific enhancer activity of E22-2
Multiple lines of evidence suggest that E22-2 is an ILC3-specific enhancer that may also have activity in some -T cells. To more directly test its activity profile, we analyzed a published transgenic mouse engineered to carry an IL-22-eGFP reporter that has only E22-234. The BAC transgene in this mouse strain has GFP inserted upstream of the Il22 coding sequences in exon 1, disrupting its signal peptide and ensuring cytoplasmic GFP expression (Supplementary Fig. 6a). With regards to regulatory regions, the BAC transgene contained ~30 kb of sequence 5’ to the Il22 promoter. As such, the BAC reporter included E22-2, but lacked E22-1, the latter of which is required for expression of endogenous IL-22 in polarized Th cells. Indeed, the BAC reporter lacking E22-1 was silent in ex vivo polarized Th22 cells, despite expression of endogenous IL-22 in ~20% of the cultured cells (Fig. 6a). In contrast, GFP was expressed in short-term cultures of primary SI-LP ILC3s (~40% of cells), even in a fraction of cells that were not expressing endogenous IL-22 (Supplementary Fig. 6b). Upon stimulation with type 3 agonists, the percentage of GFP+ cells increased (~60% of cells), and a higher proportion expressed endogenous IL-22 (Supplementary Fig. 6b, c).

In these initial experiments, ILC3s were fixed to stain for endogenous IL-22. However, fixation can result in loss of GFP fluorescence signal35. Indeed, when we directly analyzed SI-LP cell populations without fixation, 80-90% of ILC3s and ~20% of γδ-T cells were GFP+, whereas the reporter was expressed in less than 2% of the other cell types examined, including αβ-T cells (Fig. 6b). For a more comprehensive analysis, we examined reporter expression in cell populations from different primary or secondary lymphoid tissues (Supplementary Fig. 8). As shown in Fig. 6c, the E22-2-driven reporter was expressed robustly in ILC3s derived from all secondary lymphoid organs (mesenteric and peripheral lymph nodes, spleen); whereas GFP was undetectable in thymocytes, NK, B, αβ-, γδ-, or NK-T cells from these tissues. In the large intestine LP (LI-LP), we observed a low percentage of GFP+ γδ-T and NKT cells (~5%), but their proportion paled in comparison with the >70% of ILC3s expressing the reporter in this environment. Hence, in line with our enhancer knockout data, E22-2 is sufficient to drive Il22 gene expression in ILC3s and in a small percentage of γδ-T cells, but not in conventional CD4+ αβ-T cells.

To further establish E22-2 specificity during an immune response, we monitored LI-LP cells during C. rodentium infection. To also have a readout for endogenous Il22, we crossed the IL-22-eGFP transgene (E22-2 only) with the Catch22 strain, which harbors an IL-22 knock-in/knock-out reporter (TdTomato) and, as such, retains all Il22 regulatory elements36. IL-22-eGFP x Catch22 mice were euthanized at day 3 or day 13 post-infection and unfixed colon-LP cells were analyzed by flow cytometry. As expected, endogenous IL-22 expression, as measured by TdTomato, and expression of the E22-2-driven GFP reporter was largely limited to ILC3s at the early time point, but both were also detected in a very small percentage of γδ-T cells (Fig. 6d). In contrast, both reporters were silent in αβ-T cells. At the later time point, when both ILC3s and αβ-T cells are involved in the response to C. rodentium13,14, a high percentage of ILC3s expressed both reporters (Fig. 6e). Although ~20% of αβ-T cells, presumably Th17/22 cells, expressed the knock-in reporter (TdTomato), <5% expressed the E22-2-driven reporter (GFP) at 13 days post-infection. Moreover, only a low percentage of γδ-T cells expressed either of the reporters at each time point. Collectively, these data establish that the enhancer function of E22-2 is primarily restricted to ILC3s in vivo, with modest activity in a small percentage of γδ-T cells but very low to no activity in αβ-T cells. Thus, E22-2 has a rare activity profile in which its functions are restricted to the innate lymphoid counterpart of its T helper subset.

A contingent enhancer mechanism for the endogenous Il22 locus
A curious feature of enhancer profiles in the context of IL-22 expression was the absence of an additive effect in cells from the E22-1/2 versus the single knockout animals. Indeed, E22-1 mice almost perfectly phenocopied the E22-1/2 strain. One possibility is that E22-2 function in innate immune cells relies, in part, on the presence of E22-1 in their native chromosomal context; whereas, in a BAC transgene, E22-2 can function independently. To investigate this possibility, we deleted E22-1 and E22-2 independently in Mnk3 cells. Consistent with our CRISPRi data (Fig. 2d, e), both E22-1- and E22-2-deficient Mnk3 lines exhibited reduced IL-22 expression compared with their WT sister subclones, whereas IL-17F levels remained unaltered (Fig. 7a-d). To test the reciprocal impact of each enhancer deletion, we probed chromatin accessibility in the WT and mutant subclones using ATAC-seq. As shown in Fig. 7e, deletion of E22-2 had little impact on E22-1 accessibility, perhaps slightly augmenting this feature versus its WT counterpart. In contrast, deletion of E22-1 substantially attenuated, but did not abolish E22-2 accessibility.

We next probed another metric of enhancer function – promoter contact – using chromosome conformation capture (3C). Both enhancers exhibited elevated levels of Il22 promoter contacts when WT subclones were stimulated with type 3 agonists (Fig. 7f). Levels of these interactions were significantly higher than those measured for a pre-B cell line (63- 12), and contacts were undetectable between the enhancers and regions downstream of Il22 (+25 and +39), supporting the specificity of the enhancer–Il22 promoter interactions. As expected, contacts between the Il22 promoter and the regions spanning E22-1 or E22-2 were lost in the respective enhancer deletion mutants, demonstrating the enhancer dependence of these interactions. However, contacts between E22-2 and the Il22 promoter were reduced to background levels upon deletion of E22-1, but the reciprocal deletion of E22-2 had no detectable impact of E22-1 crosslinking to the Il22 promoter. Together, our data suggest that, at the endogenous Il22 locus, the full activity of E22-2 is contingent upon the presence of E22-1, which likely explains the absence of an additive impact in double mutant mice and why E22-1 phenocopy E22-1/2 mice.

Transcription factors required for E22-2 function and specificity
Our in vivo data demonstrated that E22-1 was sufficient for driving normal levels of IL-22 expression in αβ-T cells, but both enhancers were required in ILC3s. Importantly, E22-2 exhibited a rare lack of enhancer activity in αβ-T cells. To determine the core elements of each enhancer that are crucial for their distinct activity profiles, we divided them into segments (Supplementary Fig. 7a) and performed luciferase reporter assays in Mnk3. E22-1 enhancer activity is restricted to a 400 bp fragment, called E22-1b, which coincides with the core of its corresponding ATAC peak in lymphoid cells (Supplementary Fig. 7b). A prior study had shown that this region is an enhancer in cultured T cells, and it requires Runx1 and RORγt binding motifs for its activity37. Consistent with this, both of these motifs are essential for E22-1 enhancer activity in Mnk3 (Supplementary Fig. 7c). As such, we focused on E22-2, whose activity appears to be primarily restricted to ILC3s and a small percentage of gut γδ-T cells. Once again, the core ATAC peak from E22-2, which is approximately 460 bp (E22-2c), harbors all enhancer activity in Mnk3 cells (Supplementary Fig. 7b). In silico analysis of E22-2c using the JASPAR algorithm predicted binding sites for Runx, Bcl11b, RORγt and Batf/AP-1 TFs (Fig. 8a). To test their individual contributions to E22-2c enhancer function, we mutated each site and performed luciferase assays. As shown in Fig. 8b, each of the three Runx sites, a Batf site, and a Runx-Batf overlapping site in E22-2 were absolutely required for its enhancer function in this ILC3 cell line. Given their expression patterns in Mnk3 cells (Supplementary Fig. 7d, e) it seems likely that Batf, a TF also important for ILC3 functions,38,39 is responsible for induction of enhancer activity of E22-2 following exposure to type 3 agonists.

Among the members of the Runx TF family, Runx3 is necessary for ILC3 development and is expressed at the highest level in Mnk3 (Supplementary Fig. 7e) and primary ILC3s40. To test whether this factor binds to E22-2, we performed CUT&RUN using a Runx3 antibody. We obtained robust signals for Runx3 binding in Mnk3 cells at its known site in the Rorc promoter but detected no binding at a negative control site in the Gapdh promoter (Supplementary Fig. 7f)40. Importantly, Runx3 binding was detected at both ends of E22-2c, which contain sites 1-2 or sites 4-6, and these signals were more intense upon stimulation of Mnk3 cells with type 3 cytokines. In contrast, no signals were detected at any of these sites in 3T3 fibroblasts, which do not express Runx3 (Supplementary Fig. 7f). Runx3 binding to both E22-1 and E22-2 in Mnk3 was confirmed using CUT&RUN-seq (Fig. 8c). Taken together, Runx3 binding is essential for E22-2 enhancer activity in this ILC3 cell model.

We next tested whether, similar to E22-1, the predicted RORγt site was important for E22-2 enhancer activity37. Analysis of published RORγt Chip-seq data from Th17 cells41 and new CUT&RUN-seq data in Mnk3 revealed strong binding of this factor at E22-1 but minimal binding at E22-2 (Fig. 8c and Supplementary Fig. 7g). Indeed, mutation of this site in E22-2 had no significant impact on reporter expression in Mnk3 cells (Fig. 8b). Given that the RORγt site is functional in E22-1 but not in E22-2, we compared their sequences and found that the E22-1 site was a better match to the JASPAR consensus motif (Supplementary Fig. 7h). To determine the impact of the variant motifs on enhancer function, we swapped the RORγt site in E22-1b with the potentially inactive site from E22-2c. As shown in Fig. 8d, the motif swap inactivated E22-1b, confirming that the E22-2c site cannot support enhancer function in Mnk3 cells, which express the Rorc gene. Importantly, we also performed the complementary motif swap and found that replacement of the RORγt site in E22-2c with its counterpart from E22-1b endowed this chimeric enhancer with activity in primary, activated Th22 cells isolated as TdTomato+ from Catch22 mice (Fig. 8e). Likewise, this replacement also augmented E22-2c enhancer function in Mnk3 cells (Supplementary Fig. 7i), further underscoring the superior binding capability of the RORγt site from E22-1. We conclude that rather than having an ILC3-specific activation mechanism, E22-2 lacks a functional RORγt binding site, which is required for its enhancer activity in adaptive T cells. This enhancer activity profile – functional only in innate lymphoid cells – is rare among regulatory elements that control the expression of genes in Th-ILC counterparts.

Methods
Mice
All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of the Ohio State University and performed in accordance with all relevant guidelines.
All mice used in the study were on the C57BL/6J (000664, Jackson Laboratory) background. E22-1, E22-2, and E22-1/2 mice were generated using CRISPR-Cas9 technology at the Transgenic Core of the Children’s Hospital of Philadelphia Research Institute. The sgRNAs (Supplementary Data 1) flanking the E22-1 or E22-2 enhancers were designed by the GEiC core at Washington University in St. Louis. Alt-R CRISPR-Cas9 crRNAs with sgRNA sequences, Cas9 protein, and tracrRNA were purchased from Integrated DNA Technologies. Zygotes were electroporated with the ribonucleoprotein (RNP) complexes. Founder mice were screened by PCR, and the deletions were confirmed by Sanger sequencing. Founders carrying the desired deletion were then backcrossed to wild-type C57BL6/J mice for two generations, and the resulting heterozygous mice were intercrossed to generate homozygous enhancer deletion mice. For E22-1/2 mice, E22-1 was deleted from E22-2 zygotes using the same sgRNAs employed to generate E22-1 mice. The deletion of both enhancers on the same allele was confirmed by long-range PCR.
Catch22 mice36 were kindly provided by Richard M. Locksley and IL22-eGFP transgenic mice34 were purchased from the Jackson Laboratory (035005, Jackson Laboratory). For some experiments, Catch22 and IL22-eGFP mice were intercrossed to generate Catch22 x IL22-eGFP mice. All animals were bred and maintained in a specific pathogen-free animal facility at The Ohio State University, under a 12-h light/12-h dark cycle at a temperature of 20-26°C and humidity of 30-70%. Both male and female mice aged between 6-12 weeks were used. Animals within each experiment were sex- and age-matched and were either littermates or co-housed. Animals were euthanized by carbon dioxide or isoflurane anesthesia followed by cervical dislocation.

Cell lines and culture
EL4 and 63-12 cells were cultured in RPMI (Gibco, 61870-036) supplemented with 10% fetal bovine serum (Sigma, F4135), 1X Penicillin-Streptomycin (Gibco, 15140-122), and 50 µM β-mercaptoethanol (Sigma, M3148). Mnk3 cells25, were cultured in DMEM (Gibco, 11995-065) supplemented with 10% fetal bovine serum, 1X Penicillin-Streptomycin, 50 µM β-mercaptoethanol, 10 ng/ml mouse IL-7 (R&D Systems, 407-ML/CF), and 10 ng/ml mouse IL-2 (R&D Systems, 402-ML/CF). HEK293T cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1X Penicillin-Streptomycin, and 50 µM β-mercaptoethanol. For lentivirus production, HEK293T cells were co-transfected with lentiviral (1 µg), psPAX2 (1 µg), and pMD2.G (0.125 µg) plasmids using the TransIT-293 transfection reagent (Mirus, MIR 2700). Virus particles were harvested ~48 hours post-transfection and passed through a 0.45 µm syringe filter. Mnk3 cells were transduced with lentiviral supernatant in the presence of 5 µg/ml polybrene (Millipore Sigma, TR-1003-G) For experiments where sgOpti lentiviral particles were used, cells were selected for 72 hours with puromycin (1 µg/ml; Gibco, A11138-03) after ~48 hours of transduction.
For the generation of Mnk3i lines, Mnk3 cells were first transduced with rtTA3 lentiviral particles (pLenti CMV rtTA3 Blast, Addgene Plasmid #26429) and selected using blasticidin (10 µg/ml; Gibco, A1113903) after 48 hours of viral infection. After checking the expression of rtTA3 in blasticidin selected cells, we next infected them with TRE3G-dCas9-KRAB-P2A-mCherry lentiviral particles. After 48 hours of infection, cells were treated with dox (2 µg/ml; Sigma, D9891) for 24 hours, flow sorted for mcherry high cells and cloned by limiting dilution. The single cell clonal populations were screened, and a clone was selected with highest mCherry expression, a surrogate for high dCas9-KRAB expression.
E22-1 and E22-2 were deleted in Mnk3 using Cas9-sgRNA RNP complexes. Recombinant Cas9 protein (Berkeley MacroLab) was incubated with sgRNAs (1:1.2 ratio) (see Supplementary Data 1 for sgRNA sequences) for 20 minutes to assemble RNPs, which were delivered into Mnk3 cells via nucleofection using program X-005 of Nucleofector 2b System. Following recovery for 48-72 hours, cells were subjected to limiting dilution to obtain single-cell-derived subclones. Genomic deletions were confirmed by PCR and validated by Sanger sequencing.

Cloning
For Lenti-TRE3G-dCas9-KRAB-P2A-mCherry construct, tetracycline inducible TRE3G promoter was amplified from c3GIC946 (Addgene Plasmid #62191) vector adding EcoR1 restriction site at 5’ end and Mlu1 followed by BamH1 restriction enzyme sites at the 3’ end. Next, TRE3G promoter and pHR-pEF-EGFP vector47 (Addgene Plasmid #67952) were digested with EcoR1+BamH1 and ligated, resulting in pHR-TRE3G-EGFP vector. Lastly, dCas9-KRAB-P2A-mcherry cassette was swapped from pHR-SFFV-KRAB-dCas9-P2A-mCherry48 (Addgene Plasmid #60954) into pHR-TRE3G-EGFP vector from above using Mlu1 and Not1 restriction sites. sgRNAs targeting Il22 promoter, E22-1, E22-2, twin-E22-2, or scrambled sgRNA were cloned downstream of the U6 promoter in sgOpti49 (Addgene Plasmid #85681).

ATAC-seq
Nuclei were isolated from freshly harvested cells (~6x104) using ATAC lysis buffer (10 mM Tris-HCL pH 7.4, 10 mM NaCl, 3mM MgCl2, and 0.1% IGEPAL CA-630). The nuclei were then subjected to transposition reactions for 30 minutes using Tagment DNA TDE1 Enzyme (Illumina, 20034197). Purified DNA was then amplified to generate libraries using Nextera XT DNA Library Preparation Kit (Illumina, FC-131-2001). The libraries were quantified, assessed for quality and paired-end sequencing was performed with Illumina HiSeq or NovaSeq X Plus.

CRISPRi screen
The CRISPRi screen strategy was adapted from a previously published study49. ATAC- and H3K27ac-seq data from Mnk3, Mnk1, NK, ILC1, Th1, ILC3, and Th17 cells (new and published data26,27) were analyzed to identify all active/accessible regions in a 1.5 Mb space around the Il22 mouse locus (chr10:117149900-118685961, mm9 genome build). Each of the selected regions was then extended to include ±200 bp (Supplementary Data 2). The library pool was designed and cloned into sgOpti plasmid by GEiC core at Washington University. Mnk3 cells were derived from NIH-Swiss mouse, so we performed whole genome sequencing for this cell line to identify the variants when compared with the sequence of C57BL/6 genome (used for designing sgRNAs). The pool appeared to be equally effective in either mouse background. The pool consisted of 20-25 sgRNAs per regulatory region, positive controls (e.g., Il22 promoter), negative controls (ATAC negative region in chromosome 10 and promoters of certain non-essential genes such as CXCR4), and published non-targeting controls for mouse50 (Supplementary Data 2). ~82% of the above targets were covered by ≥20 sgRNAs while the remaining had fewer sgRNAs. The Mnk3i line was infected with the lentiviral library at an MOI<0.3. After 48 hours of transduction, cells were subjected to 72 hours of puromycin (1 µg/ml) selection. The selected cells were then either treated with dox (2 µg/ml) or left untreated for 24 hours and then incubated with 10 ng/ml each of IL-1 (R&D Systems, 401-ML/CF) and IL-23 (R&D Systems, 1887-ML/CF) for additional 16 hours. Cytokine-stimulated cells were treated with GolgiStop (BD Biosciences, 554724) for 6 hours and stained intracellularly with IL-22 (clone 1H8PWSR; eBioscience, 12-7221-82) and IL-17F (clone eBio18F10; eBioscience, 53-7471-82) antibodies. ~21x106 IL-17F+ cells (700-fold coverage of library) were flow sorted into IL-22-negative, -low, -medium, and -high fractions. Genomic DNA was extracted from the cellular fractions, and integrated sgRNA sequences were amplified from 19 µg genomic DNA (250-fold coverage of library) of each sample using NEBNext Ultra II Q5 (NEB, M0544S) using custom designed oligos (Supplementary Data 2). The libraries were sequenced using custom sequencing primers with Illumina HiSeq 2500. The data were analyzed using MAGeCK v0.5.7.28 Normalized count for each sample was calculated (Supplementary Data 3) and sgRNA enrichment or depletion in +dox sorted cellular populations was assessed by comparing their sgRNA abundance with corresponding -dox sorted cellular fraction.

Luciferase assay
The Il22 promoter was inserted into NheI and XhoI sites present upstream from luciferase gene of PGL3-Basic vector. The enhancer elements E22-1, E22-2, E22-1a, E22-1b, E22-1c, E22-2a, E22-2b, E22-2c, or E22-2d were cloned downstream of luciferase gene and the SV40 poly(A) signal in the PGL3-Il22 promoter construct. Mutants were created using Q5 Site-Directed Mutagenesis Kit (NEB, E0554) after designing primers (Supplementary Data 1) with the NEBaseChanger tool. Cells were resuspended in Chica buffer (5 mM KCl, 15 mM MgCl2, 0.12 M Phosphate Buffer pH 7.2, 25 mM Sodium succinate, and 25 mM Manitol) and co-nucleofected with the 5 µg of firefly luciferase and 100 ng of renilla constructs (50:1 ratio). Mnk3 cells were nucelofected using program X-005 of Nucleofector 2b System, rested for 24 hours and then either left untreated or stimulated with 10 ng/ml each of IL-1β and IL-23 overnight. EL4 cells were nucleofected using the EL4 program C-009, rested overnight and stimulated with Cell Stimulation Cocktail (eBioscience, 00 -4970-93) for 4 hours. For 63-12 cells, the X-001 program was used for nucleofection. Harvested cells were assayed in a 96-well plate using Dual-Glo Luciferase Assay System (Promega; E2920) following the manufacturer’s instructions.
Splenic naïve CD4+ T cells from T Catch22 mice were ex vivo polarized into Th22 cells and, on day 3, TdTomato positive cells were flow sorted and rested overnight in the presence of IL-2 (20 ng/ml), IL-6 (10 ng/ml; PeproTech, AF-216-16) and IL-23 (10 ng/ml). The cells were harvested, washed with 1X PBS, resuspended in P4 buffer (Lonza, V4XP-4024) and nucleofected with firefly+renilla luciferase vectors using program CM137 of 4D-Nucleofector system. The nucleofected cells were then plated in anti-CD3 and -CD28 coated plates with complete RPMI media containing IL-2 (20 ng/ml), IL-6 (10 ng/ml) and IL-23 (10 ng/ml) overnight and assayed for firefly and renilla signals as described above.

Quantitative RT-PCR (RT-qPCR):
RNA was extracted from cells using TRIzol and purified. Terminal ileum (~4 cm) or colon was cut open and rinsed with HEPES/HBSS buffer (1X HBSS, and 15 mM HEPES). Tissues (terminal ileum, colon, or skin) were homogenized in TRIzol using soft tissue homogenizing CK14 precellys lysing kit (Bertin Technologies, P000912-LYSK1-A.0). The homogenized sample was transferred to a 1.5 ml tube and chloroform added. The sample was mixed by vigorous inversion for 15 seconds, incubated at RT for 3 minutes and spun at 12000 g for 15 minutes at 4°C. The aqueous layer was transferred to columns provided with RNeasy (Qiagen) or Direct-zol (Zymo Research) Kit and proceeded for RNA isolation following manufacturer’s protocol. The isolated RNA was then reverse transcribed to cDNA using M-MuLV Reverse Transcriptase (NEB, M0253) according to the manufacturer’s protocol. qPCR was performed using SYBR Green mastermix, and gene expression was normalized to that of β-actin or Rplp2 for skin. Primers used are listed in Supplementary Data 1.

Flow cytometry
The Mnk3 lines were treated with 10 ng/ml each of IL-1β and IL-23 or left untreated overnight followed by addition of GolgiStop (BD Biosciences, 554724) for 6 hours. Cells were then intracellularly stained for IL-22 (clone 1H8PWSR; eBioscience, 12-7221-82) and IL-17F (clone eBio18F10; eBioscience, 53-7471-82) using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, 554714) following manufacturer’s protocol. Samples were acquired on Cytek Aurora or BD flow cytometers and analyzed using FlowJo software.

Tissue harvest and population analysis
Gut Lamina Propria: Small-intestines (SI) or colons were harvested and flushed to remove luminal content. Intestines were then cut open, minced into pieces and rotated in HBSS containing EDTA, FBS, and HEPES for 20 minutes, vortexed briefly and then rotated again in fresh buffer for an additional 20 minutes to remove epithelial layers, and washed thoroughly to rinse off EDTA. The tissues were then digested in complete RPMI containing Collagenase IV (100 U/ml; Sigma, C5138) for 40 minutes at 37oC. The digested tissues were passed through a 100 µm cell strainer and pelleted. Lamina propria (LP) lymphocytes were isolated using Percoll (Cytiva, 17089101) density gradient and washed with media.
For analysis of IL-22 production by SI-LP lymphocytes, the cells were treated with either 1 ng/ml or 10 ng/ml each of IL-1β and IL-23 or left untreated followed by addition of GolgiStop after 30 minutes and further incubated for 3 hours. The cells were then stained for flow cytometry analysis. SI-LP ILC3s were identified as live CD3-CD5-CD19-CD45intCD90.2hi lymphocytes and in some experiments were then further subdivided into NKp46+ CCR6-, NKp46-CCR6+, or double negative subsets. SI-LP γδ-T cells were identified as live CD19-CD45+CD3+TCRβ-TCRγ/δ+ lymphocytes.
NKT17 cells analysis: Tissue culture plates were coated for 2 hours at 37oC with anti-hamster IgG (1:50 dilution) (MP Biomedicals, 856984) followed by coating with anti-CD3 (0.25 µg/ml) (BioLegend, 100340) and anti-CD28 (1 µg/ml) (BioLegend, 102116) for 1 hour at 37oC. Cells from all peripheral (mammary, brachial, axillary, and inguinal) lymph nodes were pooled, RBCs were lysed using ACK buffer, and were cultured overnight in the above coated plates in the presence of IL-1β (10 ng/ml) and IL-23 (10 ng/ml) followed by addition of GolgiStop for additional 4 hours. Cells were first incubated with mouse CD1d PBS-57 BV421-labeled tetramer (1:100, kindly provided by NIH Tetramer Core Facility at Emory University) for 30 minutes on ice, washed twice, followed by staining for surface receptors, RORγt and cytokines (IL-22 and IL-17A) using the Foxp3 Transcription Factor Fixation/Permeabilization kit (eBioscience, 00-5521-00). NKT17 cells were identified as live lymphocytes sized CD3+TCRβ+RORγt+CD1d-PBS-57 tetramer+ population.
Ex vivo T cell polarization: Tissue culture plates were coated with anti-hamster IgG and anti-CD3+anti-CD28 as described above. Splenic naïve CD4+ T cells were MACS sorted using the naive CD4+ T Cell Isolation Kit (Miltenyi Biotec, 130-104-453) and cultured in anti-CD3/-CD28 coated plates in RPMI containing 10% FBS, 1X Glutamax, 1X Pyruvate, 1X Penicillin-Streptomycin and 50 µM β-mercaptoethanol for 3 days in either Th1 polarizing conditions: 10 ng/ml of IL-12 (R&D Systems, 419-ML/CF) and 100 ng/ml of anti-IL-4 (BioLegend, 504122) or Th22 polarizing condition: IL-6 (20 ng/ml), IL-23 (20 ng/ml), FICZ (200 nM; Sigma, SML14I89), 100 ng/ml each of anti-IL-2 (BioLegend, 503706), anti-IL-4, anti-TGFβ1,2,3 (R&D Systems, MEAB1835), and anti-IFNγ (BioLegend, 505834). For cytokine analysis, cells were stimulated with 1X Cell Stimulation Cocktail (plus protein transport inhibitors) (eBioscience, 00-4975-03) for 4 hours and stained intracellularly using IL-22 (Clone Poly5164; BioLegend, 516404) and IL-17A (Clone TC11-18H10; BD Biosciences, 560220) antibodies for flow cytometry analysis.
LIVE/DEAD Fixable Aqua Dead Cell Stain (Molecular Probes Invitrogen, L34966) or LIVE/DEAD Fixable Blue Dead Cell Stain (Molecular Probes Invitrogen, L34962) was used at 1:500 dilution to identify and exclude dead cells from the analyses.
In reporter mice analysis, cell types were identified among live lymphocytes as ILC3s: CD3-CD19-CD45intCD90.2hiCD127+, B cells: CD45+CD19+, NKT17 cells: CD19-CD45+CD3+CD1d-PBS-57 tetramer+, αβ-T cells: CD19-CD45+CD3+TCRβ+TCRγδ-CD4+, γδ-T cells: CD19-CD45+CD3+TCRβ-TCRγδ+, and NK cells: CD3-CD19-CD45+CD127-NK1.1+. Representative gating strategies shown in Supplementary Fig. 8.

Antibodies
The primary mouse cells were stained using fluorochrome conjugated antibodies against CD5 (clone 53-7.3; BioLegend, 100624), CD3 (Clone 17A2 and 145-2C11; BioLegend), CD19 (Clone 1D3/CD19; BioLegend), CD45 (Clone 30-F11; BioLegend), CD90.2 (Clone 53-2.1; BD Biosciences), CCR6 (Clone 140706; BD Biosciences, 564736), NKp46 (Clone 29A1.4; BioLegend, 137616), Streptavidin PE-Cyanine7 (eBioscience, 25-4317-82), IL-22 (Clone Poly5164; BioLegend, 516404), IL-17A (Clone TC11-18H10; BD Biosciences, 560220), IFN-γ (Clone XMG1.2; BioLegend, 505806), CD4 (Clone RM4-5; BioLegend), TCRβ (Clone H57-597; BioLegend), TCRγδ (Clone GL3; BioLegend), CD127 (Clone A7R34; BioLegend, 135043), RORγt (Clone AFKJS-9, eBiosciences, 17-6988-82), and NK1.1 (Clone PK136; BioLegend, 108707).

Citrobacter rodentium infection
After 1 week of acclimatization, 8-12 weeks age- and sex- matched mice were administered orally with 2x109 CFU of kanamycin resistant C. rodentium (DBS120 Kanr). The mice were observed and weighed daily for 20 days after infection. Fecal samples were collected, weighed, homogenized in 1X PBS and serially diluted. Different dilutions were plated on LB plates containing kanamycin. Colonies were counted after ~20-24 hours of incubation at 37oC and CFU determined as CFU/g stool = (colony count X Total volume of the sample) / (Volume plated X Dilution Factor X Wt. of the stool sample in grams).

Imiquimod-induced skin inflammation
Mice of each genotype were co-housed at least 7 days prior to the start of the experiment. The left pinnae of mice were treated topically with 31 mg of 5% imiquimod cream or a control cream (Vaseline) daily for 9 consecutive days. Fisherbrand Traceable Digital Calipers were used to measure ear thickness daily. The PASI clinical scoring system was modified from previously published report33. Briefly, a visual exam of the ears was performed daily to assess the redness, scaling, and thickness on a scale of 0-4 (0-none, 1-mild, 2-moderate, 3-marked, 4-severe) and the mean for each genotype was calculated. Mice were euthanized on day 9 and the treated ear was fixed in 4% v/v paraformaldehyde for a minimum of 24 hours followed by paraffin embedding. Five µm sections were cut with a microtome and stained with hematoxylin and eosin by the Comparative Pathology & Digital Imaging Shared Resource at The Ohio State University. Samples were then observed under light microscopy and three measurements of the epidermal thickness were taken using cellSens by Olympus LS.

CUT&RUN
For CUT&RUN51, 1x106 unfixed cells bound to activated ConA-coated magnetic beads (Bangs Laboratories, BP531) were incubated overnight at 4C with either 1:100 dilution of Runx3 monoclonal Antibody (Clone 2B3; Invitrogen, MA5-17169) or mouse IgG control antibody (Diagenode, C15400001-100) in antibody buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor, 0.025% Digitonin, 2 mM EDTA) followed by 2X wash with digitonin buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor, 0.025% Digitonin) and binding of protein A/G-MNase for 1 hour at 4C. After 2X washes with digitonin buffer, beads were washed in ice-cold low salt wash buffer (20 mM HEPES, pH 7.5, 0.5 mM Spermidine, 0.025% Digitonin) and digested using MNase digestion buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.025% Digitonin) for 5 minutes on ice. The CUT&RUN fragments were released by using stop buffer (170 mM NaCl, 20 mM EGTA, 0.025% Digitonin, 25 µg/ml Glycogen, 50 µg/ml RNase A) and incubating for 30 minutes at 37°C. Input samples were prepared by fragmenting the chromatin of 1x106 cells using bioruptor. The chromatin was precipitated, purified using phenol chloroform extraction method and Qiagen MaXtract tubes. Extracted chromatin was analyzed by qPCR and enrichment in each sample was reported as the signal relative to the total amount of input DNA. Primers used are listed in Supplementary Data 1.
CUT&RUN sequencing was performed using the CUTANA ChIC/CUT&RUN (EpiCypher, 14-1048) and the CUTANA CUT&RUN Library Prep Kit (EpiCypher, 14-1001), according to the manufacturer’s instructions. The antibodies used were: Runx3 (Clone 2B3; Invitrogen, MA5-17169), mouse IgG control (Diagenode, C15400001-100), RORγt (Abcam, ab207082), and rabbit IgG control (Diagenode, C15410206). Libraries were quantified using Qubit, assessed for fragment-size distribution on a TapeStation, and sequenced on a NovaSeq X Plus to obtain > 10 million paired-end reads per sample. The data were analyzed using the Nextflow cutandrun v3.2.2 pipeline.

Chromosome Conformation Capture (3C) assay
3C assays were performed following published protocols52,53 with the following modifications. Briefly, 107 cells were fixed using 2% formaldehyde, quenched with 0.125 M glycine, and then lysed to isolate nuclei. Chromatin was digested overnight at 37°C with 400 U Bgl II (NEB, R0144M), followed by the addition of 200 U Bgl II for an additional 4 hours. Digested chromatin was ligated at room temperature for 4 hours, reverse-crosslinked overnight at 55°C, and purified by phenol-chloroform extraction. Ligated products were quantified by TaqMan-based qPCR (oligos listed in Supplementary Data 1) using a control 3C library generated from BACs spanning the mouse Il22 and Actb loci (RP23-97O1 and RP24-227B3; BACPAC Resources Center, Redmond, Washington). The relative interaction frequencies were calculated by normalizing ligation signals to the Actb control interaction.

Statistical methods
Prism (GraphPad Software) was used to perform statistical analysis and to plot the data. The statistical tests used, and p values are indicated in the figure legends and figures. P values were calculated using either unpaired t-tests, one-way ANOVA, two-way ANOVA, or three-way ANOVA.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study by Saini et al. presents a comprehensive analysis of two conserved enhancers, E22-1 and E22-2, that regulate IL-22 expression in type 3 lymphoid cells, including ILC3s and Th17/22 cells. Their findings reveal that E22-1 is active in both innate (ILC3s) and adaptive (Th17/22) lymphoid cells, whereas E22-2 is specifically expressed in ILC3s and a subset of γδ-T cells. Mechanistically, the ILC3 specificity of E22-2 is attributed to multiple Runx3 binding sites and the absence of a functional RORγt motif, which is present in E22-1. The study employs a rigorous methodological approach, integrating CRISPRi screening, genetic knockout models, and functional assays to delineate enhancer-specific roles in homeostasis, infection, and inflammation. While the findings are compelling, several aspects require further clarification or investigation to strengthen the manuscript for publication in a peer-reviewed journal.\n1. Based on the data from Fig. 3, IL-22 production by ILC3 subsets in E22-1/2 knockout mice is reduced by 2-3 fold. However, this reduction is not as severe as the impact observed in Th22, iNKT17, and γδ-T cell subsets. This raises the question of whether the CRISPRi screening of potential cis-regulatory elements using the ILC3 cell model Mnk3 cells fully captured all regulatory elements governing IL-22 expression, particularly those specific to ILC3s. A more detailed discussion is needed to address this limitation.\n2. In Fig. 3h, i and Fig. 5, the authors use C. rodentium intestinal infection and imiquimod-induced skin inflammation mouse models to assess the impact of E22-1/2 deletion on IL-22 production by ILC3s in vivo. However, since these mice were generated using CRISPR-Cas9 technology, the potential influence of non-immune cells in the microenvironment cannot be ruled out. To confirm that the observed effects are intrinsically linked to ILC3s, it is recommended to isolate E22-1/2-deficient ILC3s and establish an adoptive transfer model.\n3. In Fig. 4, the authors observe that the loss of either E22-1 or E22-2 results in a similar reduction in IL-22 production by ILC3 and γδ-T cell subsets. They conclude that these enhancers function in a compensatory rather than synergistic manner and discuss this finding accordingly. To further investigate this, it is suggested that ATAC-seq or Hi-C sequencing be performed using the ILC3 cell model Mnk3 cells to determine whether these enhancers physically interact with the IL22 promoter independently.\n4. Based on the data from Fig. 7, the authors propose that RORγt and Runx3 are key regulators of the E22-1/2 enhancers controlling IL22 expression in ILC3s. However, direct evidence demonstrating that these transcription factors bind to the enhancers to regulate IL22 expression remains incomplete. To further clarify their roles, it is suggested to overexpress RORγt and Runx3 in both E22-1/2-deficient and wild-type ILC3 subsets and assess whether the ability to restore IL22 expression is altered.\n5. It is well established that multiple transcription factors, including AHR and STAT3, regulate IL22 expression in ILC3s, with STAT3 responding to IL-1β and IL-23 signaling to promote IL22 production. Given this, it is recommended to discuss the potential cooperative regulatory mechanisms between these transcription factors, the E22-1/2 enhancers, and Runx3 in controlling IL22 expression.", "reviewer_2": "The paper by Saini et al. identifies and characterizes two IL-22 gene enhancers, E22-1 and E22-2. Both enhancers collaboratively regulate IL-22 expression, with E22-1 displaying broad activity in Th17/22 cells and innate cells and E22-2 providing specific regulation within ILC3 cells and part of TCRγδ cells. The findings demonstrate that the coordinated action of these enhancers ensures proper IL-22 expression necessary for homeostasis, pathogen defense, and inflammatory responses.\nThis is an intriguing, technically robust study offering valuable insights into differential gene regulation mechanisms between adaptive Th17/22 cells and innate lymphoid cell counterpart, ILC3.\nHowever, the mechanism underlying the selective activity of E22-2 in ILC3 remains unclear. While the authors suggest selective activity due to critical Runx3 binding sites and a lack of RORγ binding sites, implying greater Runx3 activity in ILC3 relative to Th17/22 cells, direct evidence supporting this hypothesis is absent. Furthermore, existing literature indicates Runx3 expression in Th17 cells as well (DOI: 10.3892/mmr.2016.5108). Clarification on this point would significantly strengthen the conclusions.\nAnother aspect requiring attention pertains to the role of E22-2 in TCRγδ cells. The authors note partial functionality of E22-2 in these cells but do not specify details regarding subsets or TCR δ or γ variable gene segment usage. Although not critical to the main findings, it would be beneficial if the authors speculated in their discussion regarding potential TCRγδ subsets that utilize E22-2.", "reviewer_3": "The paper authored by Saini et al. explores the functional role of two potential enhancers, E22-1 and E22-2, in the regulation of the cytokine IL-22, particularly focusing on innate lymphoid cells type 3 (ILC3s). The data presented supports the hypothesis that both enhancers contribute to IL-22 regulation in varying contexts. While the manuscript is generally well-structured and provides substantial evidence for the authors' conclusions, several significant issues need to be addressed that could impact the overall findings of the study.\nMajor Points:\n1. A critical oversight in the paper is the lack of comprehensive examination regarding the co-production of IL-17A along with IL-22. Throughout the text, IL-17 is often referred to without specific mention of the subclass (e.g., IL-17A vs. IL-17F), which is particularly notable in Fig. 2e. The study would benefit immensely from assessing whether the enhancers E22-1 and E22-2 affect IL-17A levels. Specifically, how do endogenous IL-17A levels change in single or dual knockouts in models like c. rod or the imiquimod (IMQ) psoriasis model? Addressing this could significantly enhance the strength of the conclusions drawn.\n2. The definitions of cell types lack clarity throughout the manuscript. For instance, it's unclear whether the NK cells referenced are NK22 cells. The methods section provides insufficient detail, and the figure legends lack specificity regarding the markers used for identification. Clarifying these details will enhance the reproducibility and interpretability of the study.\n3. The manuscript acknowledges variability in the cellular landscape of IL-22 depending on the tissue examined; however, it would be beneficial to specify which exact regions of the gut are being investigated. Are the cells sourced from small intestine lamina propria (SI-LPs), mesenteric lymph nodes (mLNs), or colon LPs? The rationale for selecting different sites should be made explicit, especially considering that only SI-LPs are utilized for knockout studies shown in Figures 3 and 4. Were changes in the colon or skin context assessed? Providing this context will deepen the reader's understanding of the enhancer effects in varying environments.\n4. The statement claiming enhancers are “Rare Among Regulatory Elements” presents a compelling point that warrants further elaboration. A discussion on the rarity of these enhancers, including potential implications such as evolutionary adaptations in regulatory elements and the distinct roles based on binding site locations, could enrich the manuscript. Does this rarity suggest a nuanced mechanism for fine-tuning IL-22 expression across different innate versus adaptive contexts without relying on specific enhancer machinery?\n5. The genes IFN-gamma (IFNG), IL-22, IL-26, and IL12A (IL-12 p35 subunit) are located on chromosome 12, yet being in close proximity does not inherently imply shared regulatory elements. It is essential for the authors to discuss any data regarding the regulatory domains and potential co-regulation among these genes to provide a comprehensive understanding of their interaction.\n6. The manuscript should discuss how enhancer activity varies in different scenarios, such as inflammation versus infection, and whether these changes lead to downstream effects on IL-22 production.\n7. In the context of the knockout animals used in the study, it would be pertinent to investigate whether there are notable differences in the levels of IL-22 binding protein (IL-22-BP). This analysis could provide further insights into the functional consequences of enhancer deletions.\n8. The terminology of \"regulate\" is somewhat vague and should be refined. It is crucial to specify whether E22-1 and E22-2 act as activators or repressors of IL-22 expression. Additionally, insights into the mechanistic aspects, such as the binding of specific transcription factors, would substantially strengthen the discussion.", "reviewer_4": "Here the authors use a CRISPR based screen in the MNK3 cell line to identify regions regulating IL-22 production in response to dual IL-1 and IL-23 stimulation. The authors identify E22-1 and E22-2 and nicely show that E22-1 regulates activity in EL4 and MNK3 but E22-2 only in MNK3. The authors perform rigorous controls to show the specificity of E22-2 in regulating Il22 and ruling out potential contribution of twin E22-2. The authors subsequently develop novel mouse models to study the impact of E22-1 and 2 separately and independently. These important results demonstrate the impact of E22-2 in ILC3 and gdTs in regulating effector function in mucosal epithelium. The authors further demonstrate that Runx3 (and not Rorgt) binding is critical, establishing the mechanism for this ILC specificity. These data are very important in understanding the mechanism of cell immunity in health and disease.\nSpecific comments:\nFor the initial screening, it seems that cells were gated on IL-17F+ presumably as a positive control. Can the authors clarify this briefly in the text and maybe add the gating strategy even in supplement for clarity.\nIt is not exactly clear why the authors stimulate with both IL-1 and IL-23. Is it expected that these will go through different signaling pathways, or are they synergistic? Can the authors comment on the specificity of these findings for one pathway or the other versus simply signal strength which is convergent on this genomic region?\nFig. 3, the authors nicely show IL-22 in all subsets. Could they please show the represented flow for that as well? For example, CCR6 or NKp46 vs. IL-22.\nFigure 4 is very impressive. Did the authors test the impact of Citrobacter infection in single E22-2 deletion mice? Also, in figure 6d-e, they show the sufficiency of E22-2 to rescue ILC3 induced in Citrobacter, but the impact on clinical disease (clearance, inflammation, etc.) is not shown.\nSome minor points:\nIn fig 3 and 4, for the quantification of IL22 expression, the author used IL22 MFI but not percentage. Maybe they could show both or made it consistent with representative flow plot?\nFor the AMP genes analysis, the author used the tissue from terminal ileum. Did author detect these genes from colon tissue under steady-state or after C.rodentuim infection?"}, "author_response": "We sincerely thank the editors and reviewers for their time and efforts, providing us with constructive feedback that we have incorporated to improve our study. We have revised our manuscript to address all the comments, as detailed in the point-by-point response below.\nREVIEWER 1\n[...]\nComment 1: Based on the data from Fig. 3, IL-22 production by ILC3 subsets in E22-1/2 knockout mice is reduced by 2-3 fold. However, this reduction is not as severe as the impact observed in Th22, iNKT17, and γδ-T cell subsets. This raises the question of whether the CRISPRi screening of potential cis-regulatory elements using the ILC3 cell model Mnk3 cells fully captured all regulatory elements governing IL-22 expression, particularly those specific to ILC3s. A more detailed discussion is needed to address this limitation.\nResponse: We agree with the Reviewer that there may be additional cis-elements operating, especially in ILC3s, which the CRIPSRi screen in Mnk3 may not have captured due to redundancies or irrelevance in the cell model (e.g., the super-enhancer). We now elaborate upon this consideration further in the Results (line#119) and Discussion (line#433) sections.\nComment 2: In Fig. 3h, i and Fig. 5, the authors use C. rodentium intestinal infection and imiquimod-induced skin inflammation mouse models to assess the impact of E22-1/2 deletion on IL-22 production by ILC3s in vivo. However, since these mice were generated using CRISPR-Cas9 technology, the potential influence of non-immune cells in the microenvironment cannot be ruled out. To confirm that the observed effects are intrinsically linked to ILC3s, it is recommended to isolate E22-1/2-deficient ILC3s and establish an adoptive transfer model.\nResponse: We did not intend to imply that the phenotypes of enhancer double knockouts for C. rodentium infections or the imiquimod (IMQ) model were due to attenuation of IL-22 specifically in ILC3s. In this regard, T lineage cells almost certainly contribute to the phenotypes at later time points, and we present each result only in the general context of type 3 immune cells (ILC3s and type 3 Th cells). The C. rodentium experiment served to demonstrate that the two enhancers are important for achieving levels of IL-22 expression needed to efficiently clear the gut-associated pathogen – i.e., showing that E22-1 and E22-2 are important in vivo rather than only in the Mnk3 cell model. Because IL-22 is expressed exclusively by immune cells: ILC3, -T, Th22, and iNKT17 cells, we feel that an adoptive transfer model would not add to our conclusion.\nComment 3: In Fig. 4, the authors observe that the loss of either E22-1 or E22-2 results in a similar reduction in IL-22 production by ILC3 and γδ-T cell subsets. They conclude that these enhancers function in a compensatory rather than synergistic manner and discuss this finding accordingly. To further investigate this, it is suggested that ATAC-seq or Hi-C sequencing be performed using the ILC3 cell model Mnk3 cells to determine whether these enhancers physically interact with the IL22 promoter independently.\nResponse: We thank the Reviewer for this insightful suggestion. In the revised manuscript, we now include experiments with Mnk3 subclones in which either E22-1 or E22-2 were deleted from both Il22 alleles. ATAC and 3C assays revealed that both E22-1 and E22-2 make specific contacts with the Il22 promoter (new Fig. 7). We find that deletion of E22-2 has no effect on either E22-1 accessibility or its promoter contacts. In contrast, deletion of E22-1 significantly reduces E22-2 accessibility and promoter contacts. The implications of these data, especially the contingent activation of E22-2 at the endogenous Il22 locus, are now presented in a new Results section, as well as in the Discussion.\nComment 4: Based on the data from Fig. 7, the authors propose that RORγt and Runx3 are key regulators of the E22-1/2 enhancers controlling IL22 expression in ILC3s. However, direct evidence demonstrating that these transcription factors bind to the enhancers to regulate IL22 expression remains incomplete. To further clarify their roles, it is suggested to overexpress RORγt and Runx3 in both E22-1/2-deficient and wild-type ILC3 subsets and assess whether the ability to restore IL22 expression is altered.\nResponse: We appreciate this comment; however, we do not feel that the suggested approach would clarify the roles of RORt and Runx3 since these factors are already expressed at high levels in ILC3s, and overexpression could have numerous indirect effects. As an alternative, we directly assessed binding of these factors using CUT&RUN-seq with RORt and Runx3 antibodies in untreated or stimulated Mnk3 cells (new Fig. 8c). Consistent with our previous model, we found that Runx3 binds to both E22-1 and E22-2, while RORt binds only to E22-1 and not to the variant site in E22-2.\nIn reporter assays, we also show that mutation of the RORt site in E22-1 abolishes its enhancer function in both primary Th22 and Mnk3 cells (Figs. 8d and e in revised manuscript). Similarly, we now show that mutation of the Runx site in E22-1 substantially reduces its enhancer activity (new Fig. S7c). By comparison, only the Runx sites in E22-2 are necessary for its enhancer activity, while the predicted RORt is dispensable.\nComment 5: It is well established that multiple transcription factors, including AHR and STAT3, regulate IL22 expression in ILC3s, with STAT3 responding to IL-1β and IL-23 signaling to promote IL22 production. Given this, it is recommended to discuss the potential cooperative regulatory mechanisms between these transcription factors, the E22-1/2 enhancers, and Runx3 in controlling IL22 expression.\nResponse: Thank you for this insightful suggestion. We have now expanded the discussion to include these concepts in revised Discussion section (line#438).\nREVIEWER 2\n[...]\nResponse: We apologize that we did not make the proposed mechanism clearer in the original manuscript. To clarify, our model is that E22-2 activity is restricted to innate lymphoid cells because this enhancer lacks a functional RORt site, so it is not active in -T cells like E22-1, which has a functional RORt site. Indeed, replacement of the non-functional site in E22-2 with the functional site from E22-1 endows E22-2 with enhancer activity in primary Th22 cells (Fig. 8e in revised manuscript). Rather than ILC3s having greater Runx3 activity, we feel that the multiple Runx sites in E22-2, each of which is essential, together with an AP1 site, are sufficient to provide enhancer activity in ILC3s but not in Th cells, where RORt seems necessary.\nAlso, as mentioned above (response #4 to Reviewer 1), we have now performed CUT&RUN-seq experiments for Runx3 and RORt in Mnk3 cells. These new data show that Runx3 binds to both E22-1 and E22-2, while RORt binds only to E22-1 (new Fig. 8c). Furthermore, our new luciferase reporter assays suggest that both RORt and Runx binding motifs are necessary for the enhancer activity of E22-1 (new Fig. S7c). We have modified the Discussion to further clarify our model for factors involved in cell type specificity.\nAnother aspect requiring attention pertains to the role of E22-2 in TCRγδ cells. The authors note partial functionality of E22-2 in these cells but do not specify details regarding subsets or TCR δ or γ variable gene segment usage. Although not critical to the main findings, it would be beneficial if the authors speculated in their discussion regarding potential TCRγδ subsets that utilize E22-2.\nResponse: We thank the Reviewer for raising this point. We analyzed γδ-T cells using antibodies for gamma variant chains 1, 4, and 7 in small intestine lamina propria of IL-22-eGFP transgenic mice. Our preliminary results with 4 mice (data below) revealed no significant differences in usage of these Vγ chains for the GFP+ vs GFP- populations. We are happy to repeat this experiment and include in supplemental data should the Reviewer request it.\nIn addition, we have revised the manuscript to state that the transgene is expressed in a small percentage of γδ-T cells rather than a small subset.\nREVIEWER 3\n[...]\nResponse: We thank the Reviewer for making this important point. We now clarify in Figs. S3 and S4 that steady-state IL-17A levels were not significantly different in primary Th and NKT cells from mutant and WT mice. Because Mnk3 cells do not express detectable IL-17A (now mentioned in Results), we analyzed IL-17F instead. We have revised the manuscript and figures to specifically name the subclass of IL-17 measured in each experiment (IL-17A or Il-17F).\nIn addition, we have added new data measuring both Il17a and Il17f expression in the IMQ-induced psoriasis model. These new data reveal no significant differences among the mutant and WT mice for Il17a and Il17f expression on day 5 post IMQ treatment (new Figs. 5h and i).\nComment 2: The definitions of cell types lack clarity throughout the manuscript. For instance, it's unclear whether the NK cells referenced are NK22 cells. The methods section provides insufficient detail, and the figure legends lack specificity regarding the markers used for identification. Clarifying these details will enhance the reproducibility and interpretability of the study.\nResponse: As indicated in the Methods section, the markers used to identify NK cells were tailored for cNK cells. The markers used to identify other cell types have been detailed in the Methods section entitled, “Tissue harvest and population analysis”.\nComment 3: The manuscript acknowledges variability in the cellular landscape of IL-22 depending on the tissue examined; however, it would be beneficial to specify which exact regions of the gut are being investigated. Are the cells sourced from small intestine lamina propria (SI-LPs), mesenteric lymph nodes (mLNs), or colon LPs? The rationale for selecting different sites should be made explicit, especially considering that only SI-LPs are utilized for knockout studies shown in Figures 3 and 4. Were changes in the colon or skin context assessed? Providing this context will deepen the reader's understanding of the enhancer effects in varying environments.\nResponse: Thank you for this comment. We agree that clearly specifying the tissue sources and why we chose them is important for interpretation. In the revised manuscript, we have indicated the origin of cells in the Results and Figure legends (SI-LP, colon LP, or mLN). We have carefully re-checked the text and ensured that these designations are consistently stated throughout.\nGiven the scarcity of ILC3s in other tissues, we focused our analyses of ILC3 subsets and γδ-T cells on SI-LP in Figs. 3, 4, and 6b. Likewise, iNKT17 cells were examined in peripheral lymph nodes, where they are abundant. For Th22 cells, splenic naïve CD4+ T cells were ex vivo polarized, as few Th cells produce IL-22 under steady-state conditions. In Figs. 6c-e, to establish the cell-specificity of E22-2, we extended our analyses to other lymphoid tissues to provide a more comprehensive assessment. These rationales have now been added to the Results.\nWe also provide new analyses of Il22 transcripts in the skin during IMQ-induced psoriasis and observed a similar reduction in Il22 levels in mutant compared with WT mice (new Fig. 5g), consistent with our findings in the SI-LP.\nComment 4: The statement claiming enhancers are “Rare Among Regulatory Elements” presents a compelling point that warrants further elaboration. A discussion on the rarity of these enhancers, including potential implications such as evolutionary adaptations in regulatory elements and the distinct roles based on binding site locations, could enrich the manuscript. Does this rarity suggest a nuanced mechanism for fine-tuning IL-22 expression across different innate versus adaptive contexts without relying on specific enhancer machinery?\nResponse: We thank the Reviewer for pointing this out. In addition to the mechanism identified here for E22-2’s innate cell specificity (i.e., multiple Runx motifs and the absence of a functional RORt motif), other cell-type-specific enhancer elements have been reported recently for genes such as RORt1,2. However, the architectural features underlying their specificity remain to be defined. This has now been discussed in revised manuscript (line#455).\nComment 5: The genes IFN-gamma (IFNG), IL-22, IL-26, and IL12A (IL-12 p35 subunit) are located on chromosome 12, yet being in close proximity does not inherently imply shared regulatory elements. It is essential for the authors to discuss any data regarding the regulatory domains and potential co-regulation among these genes to provide a comprehensive understanding of their interaction.\nResponse: Indeed, both Il22 and Ifng are located on chromosome 12 in humans and chromosome 10 in mouse, whereas the gene for IL-12A is located on chromosome 3 in both human and mouse. IL-26, however, is present only in humans and is absent in mice. Previous studies have demonstrated that Ifng and Il22 reside in distinct topological domains in activated Th cells and are insulated by a CTCF-binding site, indicating that they are regulated by independent cis-regulatory elements3. In line with this, we now present data showing that deletion of E22-1/2 has no effect on IFN- expression in ex vivo polarized Th1 cells (new Fig. S3j). We have revised the Results section accordingly to incorporate these important aspects of cytokine regulation.\nComment 6: The manuscript should discuss how enhancer activity varies in different scenarios, such as inflammation versus infection, and whether these changes lead to downstream effects on IL-22 production.\nResponse: In the revised manuscript, we show that the enhancers contribute to IL-22 production and its downstream effects when challenged by inflammation (IMQ-induced psoriasis, Fig. 5), C. rodentium infection (Fig. 3), or at steady-state (AMP and IL-22-eGFP transgenic mice, Figs. 3, 4, and 6).\nComment 7: In the context of the knockout animals used in the study, it would be pertinent to investigate whether there are notable differences in the levels of IL-22 binding protein (IL-22-BP). This analysis could provide further insights into the functional consequences of enhancer deletions.\nResponse: IL-22BP is expressed mostly by dendritic cells; however, the conventional IL-22R, required for binding of IL-22 to induce downstream signaling/functions is expressed by cells of non-hematopoietic origin, such as epithelial and stromal cells. As shown here, we analyzed Il22bp expression in the colon at steady state and did not see any changes in mRNA levels between E22-1/2 mutant (purple) and WT mice (black).\nComment 8: The terminology of \"regulate\" is somewhat vague and should be refined. It is crucial to specify whether E22-1 and E22-2 act as activators or repressors of IL-22 expression. Additionally, insights into the mechanistic aspects, such as the binding of specific transcription factors, would substantially strengthen the discussion.\nResponse: We have revised the text to specify as “activate” or “augment” when referring to enhancer activity.\nREVIEWER 4\n[...]\nSpecific Comment 1: For the initial screening, it seems that cells were gated on IL-17F+ presumably as a positive control. Can the authors clarify this briefly in the text and maybe add the gating strategy even in supplement for clarity.\nResponse: We thank the Reviewer for pointing this out. Indeed, the initial screening was gated on IL-17F+ cells. This has been clarified in the Methods section. Additionally, we have now included the gating strategy in new Fig. S1a of the revised manuscript.\nSpecific Comment 2: It is not exactly clear why the authors stimulate with both IL-1 and IL-23. Is it expected that these will go through different signaling pathways, or are they synergistic? Can the authors comment on the specificity of these findings for one pathway or the other versus simply signal strength which is convergent on this genomic region?\nResponse: IL-23 serves as the primary inducer of IL-22 in type 3 immune cells, with maximal IL-22 levels achieved in the presence of IL-1 (see Introduction line#64). Therefore, the cells were stimulated with both IL-1 and IL-23 to activate synergistic pathways driving IL-22 expression.\nSpecific Comment 3: Fig. 3, the authors nicely show IL-22 in all subsets. Could they please show the represented flow for that as well? For example, CCR6 or NKp46 vs. IL-22.\nResponse: Representative flow plots for the NKp46+ subset of ILC3s were shown in Fig. 3a, but we have now added representative flow plots for CCR6+ and DN ILC3s in revised manuscript (new Figs. S3c, d).\nSpecific Comment 4: Figure 4 is very impressive. Did the authors test the impact of Citrobacter infection in single E22-2 deletion mice? Also, in figure 6d-e, they show the sufficiency of E22-2 to rescue ILC3 induced in Citrobacter, but the impact on clinical disease (clearance, inflammation, etc.) is not shown.\nResponse: We did not observe any significant difference in weight loss between E22-2 mutant and WT mice upon C. rodentium infection (data from a single experiment shown below). We did not perform clinical disease measurements on the experiments shown in Figs. 6d-e because these were performed on reporter animals, which should be expressing IL-22 from at least one wild-type allele.\nMinor Comment 1: In fig 3 and 4, for the quantification of IL22 expression, the author used IL22 MFI but not percentage. Maybe they could show both or made it consistent with representative flow plot?\nResponse: The IL-22 percentages are shown in Figs. S3 and S4.\nMinor Comment 2: For the AMP genes analysis, the author used the tissue from terminal ileum. Did author detect these genes from colon tissue under steady-state or after C. rodentuim infection?\nResponse: We thank the Reviewer for this request, and we now include new data from colon tissue at the steady-state showing that AMP genes exhibit a similar pattern of reduction as observed in the terminal ileum (new Fig. S3k)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I think the author provided a good response to the question and offered a thoughtful discussion on the article's conclusions.", "reviewer_2": "The points I had raised are adequately addressed. I have no additional comments.", "reviewer_3": "Thank you to the authors for answering all the questions I raised and altering the manuscript accordingly. No further comments.", "reviewer_4": "The authors have adequately addressed my concerns. Nice work."}, "author_response": ""}]

Decision letter