Serine 182 on Rorγt Regulates T Helper 17 and Regulatory T Cell Functions to Resolve Inflammation

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Serine 182 on RORγt regulates T helper 17 and regulatory T cell functions to resolve inflammation

Shengyun Ma1, Shefali Patel2, Nicholas Chen1, Parth R. Patel1, Benjamin S. Cho1, John

T. Chang2, Wendy Jia Men Huang1,*

1 Department of Cellular and Molecular Medicine,

University of California San Diego, La Jolla, CA 92093, U.S.A

2 Department of Medicine,

University of California San Diego, La Jolla, CA 92093, U.S.A

* Corresponding author

ABSTRACT

Unresolved inflammation causes tissue damage and contributes to autoimmune

conditions. However, the molecules and mechanisms controlling T cell-mediated

inflammation remain to be fully elucidated. Here, we report an unexpected role of the

RAR-Related Orphan Receptor-gamma protein (RORγt) in resolving tissue inflammation.

Single-cell RNA-seq (scRNA-seq) revealed that an evolutionarily conserved serine 182

residue on RORγt (RORγtS182) is critical for restricting IL-1β-mediated Th17 activities and

promoting anti-inflammatory cytokine IL-10 production in RORγt+ Treg cells in inflamed

tissues. Phospho-null RORγtS182A knock-in mice experienced delayed recovery and

succumbed to exacerbate diseases after dextran sulfate sodium (DSS) induced colitis

and experimental autoimmune encephalomyelitis (EAE) challenge. Together, these

results highlight the essential role of RORγtS182 in resolving T cell-mediated tissue

inflammation, providing a potential therapeutic target to combat autoimmune diseases.

INTRODUCTION

RAR-Related Orphan Receptor-gamma (RORγ) is an evolutionarily conserved member

of the nuclear receptor transcription factor family. The long isoform (RORγ) is broadly

expressed in liver, kidney, and muscles, and the shorter isoform (RORγt) is uniquely

found in cells of the immune system (Jetten and Joo, 2006). Both isoforms are encoded

by the RORC locus. Humans with mutations of RORC have impaired anti-bacterial and

anti-fungal immunity (Okada et al., 2015). Previous genetic studies in mouse models

identified critical roles of RORγt in preventing apoptosis of CD4+CD8+ double positive T

cells during positive selection in the thymus (Guo et al., 2016; Sun et al., 2000), facilitating

secondary lymphoid tissue organogenesis (Cherrier et al., 2012), as well as driving the

differentiation of effector T lymphocyte and innate lymphoid cells (ILCs) involved in

mucosal protection (Ciofani et al., 2012; Ivanov et al., 2006; Scoville et al., 2016). More

importantly, RORγt also regulates the effector T cell gene programs implicated in the

pathogenesis of autoimmune diseases, including the production of interleukin 17 (IL-17)

cytokines in pathogenic T helper 17 (Th17) cells and IL-10 in regulatory T cells (Tregs);

both have been shown to be dysregulated in patients with inflammatory bowel diseases

(IBD) (Fujino et al., 2003; Ivanov et al., 2006; Jiang et al., 2014; Neumann et al., 2014;

Sun et al., 2019). However, the mechanisms by which RORγt control cell-type-specific

transcription programs are not well understood, thus, limiting our ability to target this

pathway to ameliorate autoimmune problems without adversely affecting other

homeostatic processes that are also highly dependent on RORγt. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

RORγt and its transcriptional functions are regulated by post-transcriptional

modifications (PTMs), including acetylation, ubiquitination, and phosphorylation (Rutz et

al., 2016). While acetylation of RORγt by p300 impairs RORγt binding to chromatin DNA

and its target gene expression, deacetylation of RORγt mediated by SIRT1, restores the

ability of RORγt to drive Il17a expression (Lim et al., 2015). Ubiquitination at Lys69 on

RORγt is selectively required for Th17 differentiation and dispensable for T cell

development (He et al., 2017a). RORγt poly-ubiquitination and deubiquitination by E3

ubiquitin ligases (UBR5 and ITCH) and deubiquitinating enzyme A (DUBA), respectively,

define the rate of RORγt proteasomal degradation (Kathania et al., 2016; Rutz et al.,

2015). Phosphorylation of RORγt at S376 and S484 are found to stimulate and inhibit

RORγt functions in cultured Th17 cells, respectively (He et al., 2017b). In addition,

phosphorylation of RORγt at S489 promotes RORγt interaction with AhR and

subsequently enhanced IL-17A production during autoimmune responses (Chuang et al.,

2018). However, little is known about the function of those different PTMs contribute to

the diverse RORγt function in vivo, especially phosphorylation.

Here, we report that RORγt is phosphorylated at S182. Single-cell RNA-seq

(scRNA-seq) revealed that RORγtS182 not only restricts IL-1β-mediated Th17 cell effector

functions, but also promotes anti-inflammatory cytokine IL-10 production in lymphoid

tissue (LT)-like RORγt+ Treg cells in inflamed tissues. Phosphor-null RORγtS182A knock-in

mice experienced delayed recovery and succumbed to more severe disease after DSS

induced colitis and EAE challenges.

RESULTS

Serine 182 of RORγt is dispensable for T cell development in the thymus

To characterize post-translational modifications (PTMs) of RORγt, a 2xFlag-tagged

murine RORγt expression construct was transfected into HEK293ft cells. Whole-cell

lysates after transfection 48hrs were used for anti-Flag immunoprecipitation and tandem

mass-spectrometry (MS/MS) analysis. Peptide mapping to RORγt harbored

phosphorylation at S182, T197, S489, methylation at R185, and ubiquitination at K495

(Figure 1A). In particular, the serine residue in position 182 of RORγt or 203 of RORγ,

was conserved between mouse and human (Figure S1A). Phosphorylation at this site

was the most abundant among all the RORγt PTMs identified (Figure 1B), consistent with

previous reports (He et al., 2017b; Huttlin et al., 2010). To assess the in vivo function of

this serine residue, we generated phospho-null knock-in mice (RORγtS182A) by replacing

the serine codon on Rorc with that of alanine using CRISPR-Cas9 technology (Figure

1C). Heterozygous crosses yielded wildtype (WT) and homozygous RORγtS182A

littermates born in Mendelian ratio with similar growth rates (Figure S1B).

In the thymus, CD4+CD8+ thymocytes express RORγt to maintain high levels of

the anti-apoptotic factor, B-cell lymphoma-extra large (Bcl-xL). RORγt total knockout

thymocytes undergo apoptosis, resulting in the impaired generation of mature T cells (Sun

et al., 2000). In RORγtS182A CD4+CD8+ thymocytes, RORγt and Bcl-xL protein abundance

were similar to those observed in WT cells (Figure S1C-D). RORγtS182A CD4+CD8+

double-positive thymocytes matured normally into single positive CD4+ T helper and CD8+

cytotoxic T cells (Figure S1E). In the spleen, mature CD4+ T helper and CD8+ cytotoxic bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

cell populations also appeared normal in the RORγtS182A mice (Figure S1F). Together,

these results suggest that S182 of RORγt and/or its phosphorylation is dispensable for T

cell development.

scRNA-seq revealed RORγtS182 dependent Th17 and Treg populations in steady

state colon

In the intestinal lamina propria, RORγt acts as a master transcription factor regulating

differentiation and effector cytokine production in several immune cell populations,

including RORγt+Foxp3- (Th17), RORγt+Foxp3+ T cells (RORγt+ Treg), and type 3 innate

lymphoid cells (ILC3) (Cai et al., 2011; Ivanov et al., 2006; Song et al., 2015). Similar

proportion of RORγt-expressing CD4+ T cells and ILC3 (CD3-) cells were present in both

the small intestine and colon of WT and RORγtS182A mice (Figure S1G-H). Since IBDs

primarily affect the colon, we characterized the contribution of RORγtS182 to the colonic

CD4+ T cell transcriptome using scRNA-seq (10x Genomics). Colonic lamina propria

CD4+ T cells from two pairs of WT and RORγtS182A mice were captured using anti-CD4

magnetic beads (Figure 1D). From each sample, 10-13,000 cells were sequenced. A total

of 11,725 cells with Cd4>0.4 and ~1100 genes/cell were analyzed using Seraut (Stuart

et al., 2019).

UMAP (uniform manifold approximation and projection) projection revealed 12 cell

clusters with distinct gene expression patterns in our dataset. Our subsequent analysis

focused on 8175 cells (69.7% total) in clusters 0-3 and 5-6; consisting of cells with high

expression of CD4 and Cd3e (Figure 1E-F). Cells in clusters 0 and 3 had marked

expression of Il17a and Ifng, representing two distinct subsets of the Th17 lineage. Cells bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

in cluster 2 with the high level of Foxp3 and Il10 were regulatory T cells. Memory cells

(cluster 1, Cd44hi) and naive cells (cluster 5, Cd44low) had abundant expressions of Klf2,

Tcf7, and Ccr7. Proliferating CD4+ T cells (cluster 6) had high levels of Stmn1 and Ki67

(Figure 1G). At the population level, there was a significant reduction of Treg cells (cluster

2) and an increase in one of the Th17 subsets (cluster 3) in the RORγtS182A colon (Figure

1H). Results from the scRNA-seq analyses reveal an unexpected dependency of Th17

and Treg populations on RORγtS182 in the steady state of colon.

RORγtS182A augmented IL-17A production in response to IL-1

Differential gene expression analysis revealed Th17 cells in cluster 0 and cluster 3 shared

a common gene program but also harbored unique surface receptor and effector cytokine

expression patterns (Figure 2A-B). Th17 cells in cluster 0 expressed greater Il18r1, Il1rn,

Ccl2, Ccl5, Ccl7, Ccl8, Cxcl1, and Cxcl13. In contrast, Th17 cells in cluster 3 had higher

levels of Il23r, Ccr6, Il22, Ccl20, and Cxcl3. Intriguingly, Th17 cells in cluster 3 from the

RORγtS182A colon had higher expression of Il1r1 (Figure 2C). The Il1r1 gene encodes the

IL-1β receptor involves in promoting inflammatory cytokine production in Th17 cells and

contributes to pathologies in Th17-mediated autoimmunity (Coccia et al., 2012; Sutton et

al., 2006). We therefore speculated that Th17 cells in cluster 3 from the RORγtS182A colon

with higher Il1r1 expression would have enhanced Th17 cytokine production potential.

Indeed, Th17 cells in cluster 3 from the RORγtS182A colon had higher expression of Il17a

(Figure 2C).

Next, we wanted to determine whether S182 on RORγt contributes to IL-17A

production in Th17 cells downstream of IL-1β receptor signaling in a T cell-intrinsic bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

manner. Naive T cells from WT and RORγtS182A mice were purified and polarized toward

the Th17 lineage in vitro as described (Ciofani et al., 2012) (diagramed in Figure 3A). In

the presence of IL-1β, RORγtS182A Th17 cells had significantly greater IL-17A production

potential (Figure 3B and S2A). Similar RORγt protein abundance was found in WT and

RORγtS182A Th17 cells (Figure 3C). Western analysis confirmed that RORγt proteins were

phosphorylated at S182 in cultured Th17 WT cells (Figure 3D). RT-qPCR confirmed that

an increase of Il17a at the RNA level was observed in RORγtS182A Th17 cells cultured in

the presence of IL-6, IL-23, and IL-1β (Figure 3E); this culture combination is the classic

pathogenic Th17 culture condition (Ghoreschi et al., 2010). Chromatin

immunoprecipitation (ChIP-seq) experiments indicated that RORγt binding on the Il17a-

Il17f locus in WT and RORγtS182A Th17 cells were comparable (Figure 3F). However,

higher H3K27 acetylation (H3K27ac) was detected at the CNS4 regulatory region in

RORγtS182A Th17 cells (Figure 3F). Together, these results reveal the unexpected role of

RORγtS182 in negatively regulating Th17 effector cytokine production in the presence of

IL-1β (modeled in Figure 3G).

RORγtS182A T cells drive exacerbated diseases in two models of colitis

Previous reports suggest that Th17 cells generated in the presence of IL-6, IL-23, and IL-

1β contribute to autoimmune pathologies in the intestine (El-Behi et al., 2011; Ghoreschi

et al., 2010). Therefore, we hypothesized that RORγtS182A Th17 cells with

hyperresponsiveness to IL-1β receptor signaling will drive tissue inflammation. In a model

of T cell transfer colitis, Rag1-/- recipients, lacking endogenous T cells, received WT or

RORγtS182A donor CD4+ naive T cells, experienced similar weight loss between day 2-16. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

However, the weights of recipients of WT cells stabilized on day 19-31, but those with

RORγtS182A cells experienced further weight reduction (Figure 4A). RT-qPCR confirmed

the increases of Il17a and Il1r1 at the RNA level (Figure 4B). These results indicate that

the contribution of S182 on RORγt and/or its phosphorylation to colonic inflammation is

CD4+ T cell-intrinsic.

We also challenged the WT and RORγtS182A cohoused littermates with 2% DSS in

the drinking water to induce acute intestinal epithelial injury, a model with disease

pathologies similar to those observed in human ulcerative colitis patients (Chassaing et

al., 2014). In this model, scRNA-seq experiment of colonic lamina propria cells from WT

mice harvested on day 10 post DSS challenge in a workflow similar to the steady state

experiment described earlier in Figure 1D revealed that Il1b was upregulated in CD4+ T

cell-associated macrophages (cluster 4) and neutrophils (cluster 10) (Figure S4A-B). In

the T cell compartment, DSS challenged colon harbored an increase of memory (cluster

1) and Treg (cluster 2) cells, and a reduction of Th17 (cluster 3) (Figure S4C-D). The

DSS-responsive genes in Th17 cells (cluster 0 and 3 depicted in Figure S4C) were

enriched with tumor necrosis factor-alpha (TNF) and oxidative phosphorylation signatures

(Figure S4E), which are pivotal pathways previously implicated in colitis (Koelink et al.,

2020; Liu et al., 2017). Consistent with our findings in the T cell transfer colitis model,

DSS challenged RORγtS182A mice had a significant delay in weight recovery on day 8-10

as compared to their WT littermates (Figure 4C). Colons harvested from DSS-challenged

RORγtS182A mice were much shorter than those from DSS-challenged WT mice (Figure

4D). Colonic CD4+ T cells from the DSS-challenged RORγtS182A mice also harbored

elevated surface expression of IL-1 receptors (Figure S3A), implicated in pathogenic bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Th17 driven autoimmunity (Cua et al., 2003; Ronchi et al., 2016). Histological analysis

revealed significantly more infiltrating immune cells present in the colons from RORγtS182A

mice on day 10 (Figure 4E). Together, these in vivo results suggest that S182 on RORγt

and/or its phosphorylation in CD4+ T cells contribute to inflammation resolution in colitis

settings.

scRNA-seq revealed RORγtS182-dependent colonic Th17 and Treg programs during

DSS challenge

To characterize the contribution of RORγtS182A to T cell programs during the resolution

phase of colonic inflammation, we harvested colonic lamina propria cells from two pairs

of WT and RORγtS182A mice on day 10 post DSS challenge for scRNA-seq. At the

population level, fewer memory T cells (cluster 1) were found in the DSS-challenged

RORγtS182A colons as compared to WT (Figure S5A). Differential gene expression

analysis revealed greater number of genes in clusters 0-3 that were upregulated in the

DSS-challenge RORγtS182A colon (Figure S5B), suggesting a global repressive role of

RORγtS182 in colonic T cells in inflamed settings. Notably, RORγtS182A Th17 cells in cluster

0 had higher Il17a expression, and cells in cluster 3 had higher pro-inflammatory cytokine

Ccl5 expression (Figure 5A-B). Flow cytometry analysis revealed that the RORγt+Foxp3-

Th17 subset had more IL-17A producers in the DSS-challenged RORγtS182A colon (Figure

5C).

In addition to the notable expansion of IL-17A producing Th17 cells, DSS-

challenged RORγtS182A mice also had significantly fewer IL-10 producers among the

RORγt+Foxp3+ (RORγt+ Treg) cells (Figure 5C). Recent studies suggested that IL-10 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

secreted by these cells helps to limit colonic inflammation (Ohnmacht et al., 2015; Sefik

et al., 2015; Xu et al., 2018; Yang et al., 2016). To identify the Treg population with

RORγtS182-dependent IL-10 production, we further divided the Tregs, in cluster 2, into 5

subsets (Figure 5D). Subsets 0 and 4 express genes characteristics of suppressive Treg

cells, including Gzmb and Ccr1 (Figure 5E), as described in previous reports (Miragaia et

al., 2019). Subsets 2 and 3 express genes characteristics of lymphoid tissue (LT)-like

Treg, including S1pr1. Subsets 1 express genes characteristics of non-lymphoid tissues

(NLT) Treg, including Gata3 and Pdcd1. The reduction of cluster 2 cell number in the

steady state RORγtS182A colon observed in Figure 1H can be traced to the loss of LT-like

(subset 3) Tregs (Figure S6A). Upon DSS challenge, IL-10 expression in LT-like (subset

3) Tregs was significantly reduced in the RORγtS182A colon (Figure 5F-G), consistent with

our observation in Figure 5C. Altogether, these data indicate that RORγtS182 negatively

regulates effector cytokine production in Th17 cells, promotes expression of anti-

inflammation cytokine, IL-10, as well as epithelial repair factors in RORγt+ Treg cells to

facilitate inflammation resolution during colitis.

RORγtS182A mice experienced the more severe disease in EAE

Finally, we assessed whether S182 of RORγt is involved in the negative regulation of

inflammation beyond intestinal mucosal injury. Previous reports suggest that pathogenic

Th17 cells drive disease in the EAE mouse model (Thakker et al., 2007) by secreting IL-

17A to recruit CD11c+ dendritic cells (DC) and further promoting IL-17A production from

bystander TCR+ type 17 (T17) cells to fuel local inflammation (Cua et al., 2003; Ivanov

et al., 2006; Solt et al., 2011; Xiao et al., 2014); modeled in Figure 6A. When challenged bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

in our model, RORγtS182A mice had higher disease scores and experienced greater weight

loss (Figure 6B-C). Total spinal cord immune infiltrates in RORγtS182A mice had higher

levels of Cd4, Cd11c, and Il17a mRNAs (Figure 6D-E). Flow cytometry results further

confirmed a significant increase in the number of IL-17A producing Th17 (CD4+TCR-)

and T17 (CD4-TCR+) cells (Figure 6F), as well as DC (CD11c+) cells, but no change

in macrophages (CD11b+F4/80+), in the spinal cord of the RORγtS182A mice (Figure 6G).

Overall, these results demonstrate the critical role of S182 on RORγt in modulating

RORγt+ T cell functions to protect against colonic mucosal inflammation and central

nervous system autoimmune pathologies.

DISCUSSION

Our study revealed the transcriptional landscapes of colonic lamina propria CD4+ T cells

in homeostasis and during colitis at the single-cell level. In addition to the naive and

memory populations, we identified two novel subsets of Th17 and five subsets of Treg

cells with distinct cell surface receptors and effector molecule expressions. Colonic Th17

cells in cluster 3 express higher levels of inflammation-related signature genes, including

Il23r. Under steady state, mutation of S182 on RORγt resulted in an expansion of this

Th17 subset in the colon. During the inflammation resolution phase, post DSS challenge,

cells in this cluster were significantly reduced. In contrast, colonic Th17 cells in cluster 0

enriched with Il18r1, Il1rn, Ccl2, Ccl5, Ccl7, Ccl8, Cxcl1, and Cxcl13 may help to establish

chemotactic gradients to regulate trafficking of local immune cells. Interestingly, DSS-

regulated genes of Th17 cells in cluster 0 were highly enriched in TNF and NFB signaling

pathways. TNF antagonists have been the mainstay in the treatment of inflammatory bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

bowel diseases (IBD) for over 20 years (Vulliemoz et al., 2020), and the NFB pathway

is known to be activated in inflamed colonic tissue from IBD patients (Atreya et al., 2008;

Liu et al., 2017). It would be interesting for future studies to explore whether Th17 cells

subset in cluster 0 is targeted by anti-TNF therapies in human IBD.

In the RORγtS182A colon, both Th17 subsets had elevated expression of Il1r1,

resulting in augmented IL-17A production in response to IL-1β signaling. In a positive

feedforward loop, IL-17A stimulates the production of IL-1β by neutrophils and monocytes

(McGinley et al., 2020), which in turn signals on T cells to potentiate intestinal

inflammatory response and encephalomyelitis in the central nervous system (Coccia et

al., 2012; Sutton et al., 2006). As a result, RORγtS182A mice with Th17 that are

hyperresponsive to IL-1β signaling had delayed recovery when challenged with DSS-

induced gut injury and succumbed to more severe pathology during EAE. These results

highlight the novel repressive function of S182 on ROR훾t in dampening IL-17A production

in response to IL-1β signaling in Th17 cells.

During the inflammation resolution phase post-DSS challenge, we also observed

an increase of colonic Treg (cluster 2) cells. S182 on RORγt is required for maintaining

LT-like (Cluster 2 subset 3) Treg population in steady state colon and their IL-10

production potential during DSS-induced colitis. RORγt+ Tregs are abundantly found in

the intestinal mucosa and peripheral lymphoid organs (Ohnmacht et al., 2015; Sefik et al.,

2015). Secretion of IL-10 by these cells helps to protect against autoimmune diseases

(Coquet et al., 2013; Ohnmacht et al., 2015; Rutz et al., 2015; Sefik et al., 2015; Xu et al.,

2018) by dampening uncontrolled production of inflammatory cytokines, including IL-17A,

and excessive intestinal inflammation in mouse models (Guo, 2016). Impaired IL-10 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

production has been associated with severe cases of inflammatory bowel diseases

patients (Ishizuka et al., 2001; Saxena et al., 2015), and several studies suggest that

administration of IL-10 in these patients as a potential therapy (Ishizuka et al., 2001;

Saxena et al., 2015). In DSS-treated RORγtS182A mice, IL-10 producing LT-like Tregs had

increased expression of S1pr4, which is involved in cell migration (Olesch et al., 2017),

as well as decreased levels of Cd44 and Id2, encoding molecules implicated in Treg

activation and IL-10 production (Bollyky et al., 2009; Hwang et al., 2018). Future studies

will be needed to investigate whether RORγtS182-dependent IL-10 expressions in LT-like

Tregs exert paracrine effects on local Th17 effector programs and contribute to the

delayed DSS and EAE disease recovery observed in RORγtS182A mice. Furthermore, it

remained to be explored whether the same or distinct kinase(s) and/or pathways are

involved in phosphorylating S182 on RORγt in these different T cell subsets.

Given the essential role of RORγt in the differentiation and function of Th17 cells

implicated in autoimmunity, many RORγt inhibitors have been developed for therapeutic

purposes. Most current inhibitors are designed to disrupt RORγt-dependent transcription

by preventing its interaction with steroid receptor coactivators. Unfortunately, as this is a

common mechanism RORγt employs across the different cell and tissue types, this

approach will likely exert undesired side-effects on thymic development and other innate

immune cells expressing RORγt. Our discovery of the unique involvement of S182 on

RORγt in regulating colonic mucosal Th17 and Treg populations and functions, but

dispensable for thymocyte development, provide a new cell-type-specific venue for

designing better therapies to combat T cell-mediated inflammatory diseases.

FIGURE LEGEND

Figure 1. scRNA-seq revealed RORγtS182-dependent Th17 and Treg populations in

steady state colon.

A. Proportion of modified and unmodified peptides identified by Tandem MS/MS

mapping to murine RORγt from whole cell lysates of 293ft cells transfected with

Flag2x-mRORγt expression construct for 48hrs.

B. Top: Major phosphorylation sites in relation to the activation function (AF-1), DNA-

binding domain (DBD), and ligand-binding domain (LBD) of RORγt. Bottom:

representative mass spectral map of one murine RORγt peptide carrying a

phosphorylated S182 residue.

C. CRISPR-Cas mediated genomic mutations to generate the RORγtS182A knock-in

mice.

D. scRNA-seq experiment workflow: CD4+ and their associated colonic lamina propria

cells were enriched with anti-CD4 microbeads. 10X Genomics droplet-based 3′

scRNA-seq was employed to reveal captured cell transcript profiles.

E. UMAP plot of twelve immune cell clusters obtained from D (left) and the average

expression of Cd3e in each cell (right).

F. Heatmap of average expression of genes with cluster-specific expression patterns

from E.

G. Violin plots of selected genes from each cluster, with gene expression scaled on the

y-axis.

H. Proportions of colonic CD4+ T cells in each cluster from two pairs of WT and

RORγtS182A littermates. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2. scRNA-seq identified two subsets of Th17 cells in steady state colon.

A. Left: zoom in view of the UMAP plot showing two neighboring populations of colonic

Th17 cells in red (cluster 0) and green (cluster 3). Right: select cell surface

receptors and effector molecules enriched in the two Th17 subsets from WT mice.

B. Heatmap of mean scaled average expression of cluster-specific genes in steady

colonic Th17 (cluster 0 and 3) and Treg (cluster 2) cells of WT and RORγtS182A.

C. Expression of Il17a and Il1r1 in colonic Th17 cell subsets (cluster 0 and 3) in the

steady state WT and RORγtS182A mice. UMAP expression plots (left) and violin plots

(right).

Figure 3. IL-1 signaling promoted dysregulated effector cytokine production in

RORγtS182A Th17 cells.

A. Th17 culture workflow in vitro.

B. Proportion of IL-17A+IL-17F+ producers among Th17 cells cultured for 3 days under

conditions as indicated. Each dot represents result from one mouse. * p-value<0.05,

** p-value<0.01 (paired t-test).

C. Geometric mean fluorescence (gMFI) of RORγt in WT and RORγtS182A Th17 cells

cultured under different conditions. Each dot represents result from one mouse.

D. Western blot analysis of total RORγt and S182-phosphorylated RORγt in cultured

Th17 cells from two pairs of WT and RORγtS182A littermates.

E. Normalized Il17a mRNA expression in WT and RORγtS182A Th17 cells cultured in

IL-6, IL-1β and IL-23 for 3 days, detected by qRT-PCR. Each dot represents result

from one mouse. * p-value<0.05 (paired t-test). bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

F. Top: IGV browser showing RORγt and p300 occupancy at Il17a-Il17f locus as

reported previously (Ciofani et al., 2012) along with the location of ChIP-qPCR

primers designed to capture each of the conserved non-coding regulatory elements

(CNS). Bottom: Enrichment of RORγt and H3K27ac at the Il17a-Il17f locus in WT

and RORγtS182A Th17 cells as determined by ChIP-qPCR. n=4, ** p<0.01 (t-test).

G. Working model: IL-1β signaling promotes dysregulated effector cytokines

production in RORγtS182A Th17 cells.

Figure 4. RORγtS182A T cells drived exacerbated diseases in two models of colitis.

A. Weight changes of Rag1-/- mice receiving WT or RORγtS182A naive CD4+ T cells.

Each dot represents result from one mouse. ** p-value<0.01 and *** p-value<0.001

(multiple t-test).

B. Expression level of Il1r1, Il23r and Il17a in colonic lamina propria of Rag1-/- mice

receiving WT or RORγtS182A naive CD4+ T cells. Each dot represents result from

one mouse. * p-value<0.05 (t-test).

C. Weight changes of WT and RORγtS182A mice challenged with 2% DSS in drinking

water for 7 days and monitored for another 3 days. Each dot represents result from

one mouse. * p-value<0.05 (multiple t-test).

D. Top: representative bright field images of colons from C. Bottom: summarized

colonic lengths of DSS treated WT and RORγtS182A mice from C harvested on day

10. Each dot represents result from one mouse. ** p-value<0.01 (t-test). bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

E. Top: representative colonic sections from D. Bottom: summarized score of colonic

inflammatory infiltrates (bottom) in DSS treated WT and RORγtS182A mice. Each

dot represents result from one mouse. * p-value<0.05 (t-test).

Figure 5. scRNA-seq revealed RORγtS182-dependent colonic Th17 and Treg

programs during DSS challenge.

S182 A. Heatmap of Log2 fold change of select RORγt -dependent genes expression in

Th17 (cluster 0 and 3) and Treg (cluster 2) from steady state and DSS challenged

colons.

B. Violin plots of the selected gene expressions in Th17 (cluster 0 and 3) and Treg

(cluster 2) cells.

C. Top: representative flow cytometry analysis. Bottom: proportion of IL-10 and IL-

17A production potential in colonic RORγt+Foxp3- (Th17), RORγt+Foxp3+ (RORγt+

Treg), RORγt-Foxp3+ (Treg), and RORγt-Foxp3- cells from DSS treated WT and

RORγtS182A mice and harvested on day 10. Each dot represents result from one

mouse. * p-value<0.05, *** p-value<0.001 (t-test).

D. Closed up UMAP of the five Treg subsets within Cluster 2.

E. Heatmap of mean scaled average expression of select Treg subsets enriched

genes from D.

F. Percentage of cells expressing RORγtS182-dependent genes (grey, p-Value<0.05)

in colonic Treg subsets 0 and 3 from DSS challenged WT and RORγtS182A mice.

Select genes were labeled and highlighted in blue. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

G. Left: UMAP displaying Il10 expression in scRNA-seq dataset. Right: violin plots of

the Il10 expression in LT-like Treg (subset 3) cells from control or DSS challenged

WT and RORγtS182A mice.

Figure 6. RORγtS182A mice experienced more severe disease in EAE model.

A. Working hypothesis: RORγtS182 acts as the upstream negative regulator of

pathogenic Th17 cell activities in EAE.

B. Disease score of MOG-immunized WT (n=7) and RORγtS182A (n=14) mice. Results

were combined of three independent experiments. ** p-value<0.01, *** p-

value<0.001 (multiple t-test).

C. Weight change of MOG-immunized WT and RORγtS182A mice. Results were

combined of three independent experiments. * p-value<0.05, *** p-value<0.001

(multiple t-test).

D. Normalized mRNA expression of Cd4, Cd11c, Cd19 and Cd11b in spinal cords

infiltrated at the peak of EAE disease (day 16-19) from MOG-immunized WT and

RORγtS182A mice as detected by qRT-PCR. Each dot represents result from one

mouse. * p-value<0.05 (paired t-test).

E. Normalized mRNA expression of the indicated cytokine genes in spinal cords

infiltrated at the peak of EAE disease (day16-19) from MOG-immunized WT and

RORγtS182A EAE mice as detected by qRT-PCR. Each dot represents result from

one mouse. * p-value<0.05 (paired t-test). bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

F. Number of total T helper (CD4+) and TCR+ (left) and IL-17A+ expressing Th17

and T17 cells (right) in EAE mice harvested at the peak of disease (day16-19).

Each dot represents result from one mouse. * p-value<0.05 (t-test).

G. CD11b+F4/80+ (macrophage, left) and CD3e-CD11c+ (DC, right) cells numbers in

the spinal cords of EAE mice harvested at the peak of disease (day16-19). Each

dot represents result from one mouse. * p-value<0.05 (t-test).

Supplementary Figure S1. Normal thymic T cell development and intestine

homeostasis in RORγtS182A mice.

A. S182 (black line) localizing to the hinge region of RORγt is conserved between

mouse and human.

B. Weight of WT (n=49) and RORγtS182A (n=57) mice assessed at the indicated ages.

Each dot represents result from one mouse.

C. Geometric mean fluorescent intensity (GMFI) of RORγt in thymic CD4+CD8+

double positive (DP) cells from WT and RORγtS182A cohoused littermates. Each dot

represents result from one mouse.

D. Representative histogram of Bcl-xL expression in thymic DP cells from WT and

RORγtS182A mice. This experiment was repeated three times on independent

biological samples with similar results.

E. Representative flow cytometry analysis of CD4 and CD8a expression in thymic cells

from WT and RORγtS182A mice. This experiment was repeated three times on

independent biological samples with similar results. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

F. Representative flow cytometry analysis of CD4 and CD8 expression in

splenocytes from WT and RORγtS182A mice. This experiment was repeated three

times on independent biological samples with similar results.

G. Proportion of RORγt+Foxp3-, RORγt+Foxp3+, and RORγt-Foxp3+ T cell subsets and

CD3e-RORγt+Foxp3- (ILC3) cells in the steady state small intestinal lamina propria

of WT (n=6) and RORγtS182A mice (n=6).

H. Proportion of RORγt+Foxp3-, RORγt+Foxp3+, and RORγt-Foxp3+ T cell subsets and

CD3e-RORγt+Foxp3- ILC3 cells in the steady state colonic lamina propria of WT

(n=6) and RORγtS182A mice (n=6).

Supplementary Figure S2. Representative cytokine production potential in Th17 cells

cultured from WT and RORγtS182A mice.

A. Representative flow cytometry analysis of IL-17A and IL-17F expression in cultured

Th17 cells from WT and RORγtS182A mice as described in Figure 3B.

Supplementary Figure S3. Cell surface IL-1R expression on colonic Th17 cells in

steady state and DSS-challenged WT and RORγtS182A mice.

A. Summary of proportions of IL-1R+ Th17 cells in colonic lamina propria of WT and

RORγtS182A mice treated with or without DSS (left) and representative flow plots

(right). Each dot represents result from one mouse. * p-value<0.05 (paired t-test).

Supplementary Figure S4. DSS-induced epithelial injury altered colonic immune cell

populations and their transcription programs in WT mice. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A. UMAP plot of colonic lamina propria cells obtained from steady state (blue) and day

10 post DSS challenged (red) WT mice.

B. Left: violin plot of select genes enriched in CD4+ T cells associated macrophages

(cluster 4) and neutrophils (cluster 10). Right: average expression of Il1b in

macrophages and neutrophiles from control and DSS challenged WT mice.

C. Altered proportions of each CD4+ T cell cluster in colonic lamina propria cells from

steady state (n=2) and day 10 post DSS treated WT mice (n=2).

D. DSS-dependent genes (p-value<0.05) identified in each cell cluster.

E. Heatmap of -Log10 p-Value of the top seven KEGG pathways from DSS-dependent

genes in clusters 0,1, 2, 3, and 5.

Supplementary Figure S5. RORγtS182-dependent cell populations and target genes in

colon under steady state and during DSS challenge.

A. Proportions of individual scRNA-seq clusters among colonic lamina propria CD4+

T cells from DSS-challenged WT and RORγtS182A mice. * p-value<0.05, n=2.

B. Number of RORγtS182-dependent genes in individual scRNA-seq clusters from

steady state (control) (left) and DSS-challenged (right) WT and RORγtS182A mice

(n=2). # of DE genes means number of differentially expressed genes.

Supplementary Figure S6. Cluster 2 Treg subsets in control and DSS challenged WT

and RORγtS182A mice

A. Proportions of Treg subclusters from steady state (top) and DSS-treated (bottom)

colons of WT (n=2) and RORγtS182A mice (n=2). * p-value<0.05 (multiple t-test). bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

METHODS

Mice

RORγtS182A were generated by CRISPR-Cas9 technology in the C57BL/6 (Jackson

Laboratories) background mice and confirmed by sanger sequencing of the Rorc locus.

Heterozygous mice were bred to yield 8-12 WT and homogenous knock-in (RORγtS182A)

cohoused littermates for paired experiments. Rag1-/- (Jackson Laboratory stock No:

002216) were obtained from Dr. John Chang’s Laboratory. Adult mice at least eight weeks

old were used. All animal studies were approved and followed the Institutional Animal

Care and Use Guidelines of the University of California San Diego.

Th17 cell culture

Mouse naive T cells were purified from spleens and lymph nodes of 8-12 weeks old mice

using the Naive CD4+ T Cell Isolation Kit according to the manufacturer’s instructions

(Miltenyi Biotec). Cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM,

Sigma Aldrich) supplemented with 10% heat-inactivated FBS (Peak Serum), 50U/50 ug

penicillin-streptomycin (Life Technologies), 2 mM glutamine (Life Technologies), and 50

μM β-mercaptoethanol (Sigma Aldrich). For polarized Th17 cell polarization, naive cells

were seeded in 24-well or 96-well plates, pre-coated with rabbit anti-hamster IgG, and

cultured in the presence of 0.25 μg/mL anti-CD3ε (eBioscience), 1 μg/mL anti-CD28

(eBioscience), 20 ng/mL IL-6 (R&D Systems), and/or 0.1 ng/mL TGF-β (R&D Systems),

20 ng/mL IL-1 (R&D Systems), 25 ng/mL IL-23 (R&D Systems) for 72 hours.

DSS induced and T cell transfer colitis

Dextran Sulfate Sodium Salt (DSS) Colitis Grade 36,000-59,000MW (MP Biomedicals)

was added to the drinking water at a final concentration of 2% (wt/vol) and administered

for 7 days. Mice were weighed every other day. On day 10, colons were collected for H&E

staining and lamina propria cells were isolation as described (Lefrancois and Lycke, 2001).

Cells were kept for RNA isolation or flow cytometry. The H&E slides from each sample

were scored in a double-blind fashion as described previously (Abbasi et al., 2020). For

T cell transfer model of colitis, 0.5 million naive CD4+ T cells isolated from mouse

splenocytes using the Naive CD4+ T Cell Isolation Kit (Miltenyi), as described above, were

injected intraperitoneally into Rag1-/- recipients. Mice weights were measured twice a

week. Pathology scoring of distal colons from DSS-challenged mice were performed blind

following previously published guidelines (Koelink et al., 2018).

scRNA-seq and analysis

Colonic lamina propria cells from DSS or non-DSS treated mice were collected and

enriched for CD4+ T cells using the mouse CD4+ T cell Isolation Kit (Miltenyi). Enriched

CD4+ cells (~10,000 per mouse) were prepared for single cell libraries using the

Chromium Single Cell 3' Reagent Kit (10xGenomics). The pooled libraries of each sample

(20,000 reads/cell) were sequenced on one lane of NovaSeq S4 following manufacturer’s

recommendations.

Cellranger v3.1.0 was used to filter, align, and count reads mapped to the mouse

reference genome (mm10-3.0.0). The Unique Molecular Identifiers (UMI) count matrix

obtained was used for downstream analysis using Seurat (v4.0.1). The cells with bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

mitochondrial counts >5%, as well as outlier cells in the top and bottom 0.2% of the total

gene number detected index were excluded. After filtering, randomly selected 10,000

cells per sample were chosen for downstream analysis. Cells with Cd4 expression lower

than 0.4 were removed, resulting in 27,420 total cells from eight samples. These cells

were scaled and normalized using log-transformation, and the top 3,000 genes were

selected for principal component analysis. The dimensions determined from PCA were

used for clustering and non-linear dimensional reduction visualizations (UMAP).

Differentially expressed genes identified by FindMarkers were used to characterize each

cell cluster. Other visualization methods from Seurat such as VlnPlot, FeaturePlot, and

DimPlot were also used.

EAE model

EAE was induced in 8-week-old mice by subcutaneous immunization with 100 μg myelin

oligodendrocyte glycoprotein (MOG35–55) peptide (GenScript Biotech) emulsified in

complete Freund’s adjuvant (CFA, Sigma-Aldrich), followed by administration of 400 ng

pertussis toxin (PTX, Sigma-Aldrich) on days 0 and 2 as described in (Bittner et al., 2014).

Clinical signs of EAE were assessed as follows: 0, no clinical signs; 1, partially limp tail;

2, paralyzed tail; 3, hind limb paresis; 4, one hind limb paralyzed; 5, both hind limbs

paralyzed; 6, hind limbs paralyzed, weakness in forelimbs; 7, hind limbs paralyzed, one

forelimb paralyzed; 8, hind limb paralyzed, both forelimbs paralyzed; 9, moribund; 10,

death.

Flow cytometry bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Cells were stimulated with 5 ng/mL Phorbol 12-myristate 13-acetate (PMA, Millipore

Sigma) and 500ng/mL ionomycin (Millipore Sigma) in the presence of GoligiStop (BD

Bioscience) for 5 hours at 37˚C, followed by cell surface marker staining.

Fixation/Permeabilization buffers (eBioscience) were used per manufacturer instructions

to assess intracellular transcription factor and cytokine expression. Antibodies are listed

in Table S1.

cDNA synthesis, qRT-PCR, and RT-PCR

Total RNA was extracted with the RNeasy kit (QIAGEN) and reverse transcribed using

iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories). Real time RT-PCR was

performed using iTaq™ Universal SYBR® Green Supermix (Bio-Rad Laboratories).

Expression data was normalized to Gapdh mRNA levels. Primer sequences are listed in

Table S2.

Chromatin immunoprecipitation

ChIP was performed on 5-10 million Th17 cells crosslinked with 1% formaldehyde.

Chromatin was sonicated and immunoprecipitated using antibodies listed in Table S1 and

captured on Dynabeads (ThermoFisher Scientific). Immunoprecipitated protein-DNA

complexes were reverse cross-linked and chromatin DNA purified as described in

(Kaikkonen et al., 2013). ChIP primer sequences are listed in Table S2.

Statistical analysis bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

All values are presented as means ± SD. Significant differences were evaluated using

GraphPad Prism 8 software. The Student’s t-test or paired t-test were used to determine

significant differences between two groups. A two-tailed p-value of <0.05 was considered

statistically significant in all experiments.

AUTHOR CONTRIBUTIONS

S.M. designed and performed in vivo, in vitro and scRNA-seq studies with the help of N.C.

scRNA-seq study was analyzed by S.P. with input from J.T.C and W.J.M.H. S.M. and

B.S.C. completed the DSS colitis studies. P.R.P. completed the double-blinded histology

scoring of the colonic sections from DSS challenged mice. J.T.C. supervised the scRNA-

seq analysis by S.P., contributed resources, and edited the manuscript. W.J.M.H. wrote

the manuscript together with S.M.

ACKNOWLEDGEMENTS

S.M., N.C., B.S.C., P.R.P, and W.J.M.H. were partially funded by the Edward Mallinckrodt,

Jr. Foundation and the National Institutes of Health (NIH) (R01 GM124494 to WJM

Huang). Illumina sequencing was conducted at the IGM Genomics Center, University of

California San Diego, with support from NIH (S10 OD026929). The Moores Cancer

Center Histology Core conducted colonic tissue sectioning and staining with support from

NIH (P30 CA23100). We thank Karen Sykes for suggestions to the manuscript.

CONFLICT OF INTEREST

All authors declare no competing financial interests. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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Serine 182 on RORγt regulates T helper 17 and regulatory T cell functions to resolve inflammation

Graphical Abstract

Inflammation Resolution

Inflammatory Th17 Serine 182 on ROR�t limits T cell mediated IL-1β Receptor intestine and central

p-RORgtS182 nervous system inflammation. IL-17 Clusters umap DSS umap Genotype umap

0 DSS RORgt 1 No DSS WT 2 5 5 3 5 4 5 6 LT-like 7 8 0 0 9 0 Treg 10 11 UMAP_2 UMAP_2 UMAP_2 − 5 − 5 − 5 p-RORgtS182 − 10 − 10 − 10 Foxp3 IL-10 − 15 − 15 − 15 −5 0 5 10 15 20 −5 0 5 10 15 20 −5 0 5 10 15 20

UMAP_1 UMAP_1 UMAP_1

Highlights • Single-cell RNA-seq of the colonic lamina propria reveals CD4+ T cell heterogeneity under steady state and during dextran sulfate induced colitis.

• The master transcription factor RORγt of Th17 and Treg cell functions is phosphorylated at serine 182.

• Serine 182 on ROR�t represses IL-1β-induced IL-17A production in in Th17 cells and potentiates IL-10 production in colonic LT-like Treg cells.

• Phospho-null ROR�tS182A mice succumb to exacerbate inflammation when challenged in models of colitis and experimental autoimmune encephalomyelitis. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

11 1212.5314 606.7693 1195.5048 598.2560 1194.5208 597.7640 E 762.3417 381.6745 745.3151 373.1612 744.3311 372.6692 6 12 1375.5947 688.3010 1358.5681 679.7877 1357.5841 679.2957 Y 633.2991 317.1532 616.2726 308.6399 615.2885 308.1479 5 13 1444.6162 722.8117 1427.5896 714.2984S1821426.6056 713.8064 S 470.2358 235.6215 453.2092 227.1082 452.2252 226.6162 4 Fig 1. scRNA-seq revealed14 1541.6689 771.3381 ROR1524.6424 762.8248gt 1523.6584dependent762.3328 P 401.2143 201.1108 384.1878Th17192.5975 and383.2037 192.1055 Treg3 15 1670.7115 835.8594 1653.6850 827.3461 1652.7009 826.8541 E 304.1615 152.5844 287.1350 144.0711 286.1510 143.5791 2 populations in steady16 state colon . R 175.1190 88.0631 158.0924 79.5498 1

A. B. C.

293ft cells: Flag2x-mRORgt mRORMascot Search�t Results PS182 pY195 PS489 Peptide View

MS/MS Fragmentation of TEVQGASCHLEYSPER Found in tr|D3YUU7|D3YUU7_MOUSEmROR� in UniProtCP_mouse, Nuclear receptor ROR-gamma OS=Mus musculus GN=Rorc PE=3 SV=1 IP: Flag à Mass spectrometry Match to Query 10743: 1941.784912 from(971.899732,2+) intensity(1313443.0000)PS203 pY216 PS510 Title: D120817_025.16159.16159.2NCBI BLAST search of TEVQGASCHLEYSPER Data ﬁle D120817_025.mgf(Parameters: blastp, nr protein database, expect=20000, no ﬁlter, PAM30) Click mouse within plot area to zoom in by factor of two about that point Or, Plot fromOther AF100 BLAST- to1 1600 web Da Fullgateways rangeDBD LBD Label all possible matches Label matches used for scoring 50 View spectrum in original style View spectrum with detailed annotation Modified peptide Show Y-axisAll matches to this query XhoI silent mutation Ser>Ala 40 Unmodified peptide Score Mr(calc) Delta Sequence Site Analysis 77.5 1941.7928 -0.0079 TEVQGASCHLEYSPER Phospho S13 100.00% 27.8 1941.7928 -0.0079 TEVQGASCHLEYSPER Phospho S7 0.00% WT 30 15.4 1941.7928 -0.0079 TEVQGASCHLEYSPER Phospho Y12 0.00% ROR�t CTT GAG TAT AGT 3.7 1941.7638 0.0211 ETVYGCFQCSVDGVK Leu Glu Tyr Ser 20 1.7 1941.7928 -0.0079 TEVQGASCHLEYSPER Phospho T1 0.00% 1.0 1941.7672 0.0177 FTIGKECEMQTMGGK ROR�tS182A CTC GAG TAT GCT Peptide count 1.0 1941.7672 0.0177 FTIGKECEMQTMGGK 10 0.4 1941.7944 -0.0095 MTELYQSLADLNNVR Leu Glu Tyr Ala

0 Mascot: http://www.matrixscience.com/

p-S182 p-T197 p-S489 u-K495 m-R185 Monoisotopic mass of neutral peptide Mr(calc): 1941.7928 Fixed modifications: Carbamidomethyl (C) (apply to specified residues or termini only) Variable modifications: S13 : Phospho (ST), with neutral losses 97.9769(shown in table), 0.0000 Ions Score: 78 Expect: 1.2e-07 Matches : 21/262 fragment ions using 25 most intense peaks (help)

# b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ # D. 1 102.0550 51.5311 84.0444 42.5258E.T 16 2 231.0975 116.0524 213.0870 107.0471 E 1743.7755 872.3914 1726.7490 863.8781 1725.7649 863.3861 15 3 330.1660 165.5866 312.1554 156.5813 V 1614.7329 807.8701 1597.7064 799.3568 1596.7224 798.8648 14 1. Colon Lamina 4 458.2245+229.6159 441.1980 221.1026 440.2140 220.6106 Q 1515.6645 758.3359 1498.6380 749.8226 1497.6539 749.3306 13 2. CD45 515.2460 258.1266enrichment498.2195 249.6134 497.2354 249.1214 G 1387.6059 694.3066 1370.5794 685.7933 1369.5954 685.3013 12 Cd3e Propria Isolation 6 586.2831 293.6452 569.2566 285.1319 568.2726 284.6399 A 1330.5845 665.7959 1313.5579 657.2826 1312.5739 656.7906 11 7 673.3151 337.1612 656.2886 328.6479 655.3046 328.1559 S 1259.5473 630.2773 1242.5208 621.7640 1241.5368 621.2720 10 8 833.3458 417.1765 816.3192 408.6633 815.3352 408.1713 C 1172.5153 586.7613 1155.4888 578.2480 1154.5048 577.7560 9 9 970.4047 485.7060 953.3782 477.1927 952.3941 476.7007 H 1012.4847 506.7460 995.4581 498.2327 994.4741 497.7407 8 RORgtWT 10 1083.4888 542.2480 1066.4622 533.7347 1065.4782 533.2427 L 875.4258 438.2165 858.3992 429.7032 857.4152 429.2112 7 (n=2) RORgtS182A (n=2) 3. scRNA-seq

TruSeq 10x Read 1 Barcode UMI Poly(dT) F. G. H. Steady state colon scRNAseq Cd44 Cblb 40 WT Control_WT Klf2 RORgt Tbc1d4 ✱ S182A 30 RORgt Control_KI Tcf7 level Expression Ccr7

Ermn 20 Mean scaled exp scaled Mean Il17a ✱ Ifng Foxp3 10 Il10 % of lamina propria cells Stmn1 Mki67 0 0 3 2 5 1 6 Clusters: 0 3 2 5 1 6 Clusters: 0 3 2 5 1 6 Clusters: 0 3 2 5 1 6 Clusters bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig 2. scRNA-seq identified two subsets of Th17 cells in the steady state colon.

A. Th17: Cluster 0 vs Cluster 3

-1 fold change: Avg Exp Avg fold change: 2

Log -2 Il22 Il23r Il1r1 Il1rn Ccl5 Ccl8 Ccl2 Ccl7 Lyz2 Ccr6 Il17a Saa3 Cxcl3 Cxcl1 Il18r1 Ccl20 Ifngr1 Cxcl16 Cxcl13 Mmp14

Steady state colon B. Cluster: 0 3 2 C. S182A S182A S182A t t t g g g WT ROR WT ROR WT ROR

Il17a Il17a Effectors 6 Cluster 0 4

Cluster 3 2

0 Transcription factors Il1r1 3 Il1r1 Expression Level Expression 2 Cluster 0 1 Cluster 3 0 Signaling mol. RORgt: WT S182A WT S182A Cluster 0 Cluster 3

Mean scaled exp bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig 3. IL-1b signaling promoted dysregulated effector cytokine production in RORgtS182A Th17 cells. IL-17A+IL-17F+ S203A weight IL-17A+IL-17F+ A. B. 80 40 WT WT ✱✱ RORgt 60 + + ✱ t t γ RORRORgtγS182AtS203A g 30 40

(72hrs) ROR Cytokines % of ROR 20 of % 20 Naive Th17 T

Weight (g) Anti-CD3 0 10 Anti-CD28 β β β IL-6

0 IL-6+TGF IL-6+IL-1 IL-6+IL-23 + 54 60 64 68 70 73 78 85 91 98 Gate: RORγt IL-6+IL-23+IL-1 109 114 119 131 141 150 C. D. E. Il17a mRNA 2000 Pair 1 Pair 2 Il17a mRNA Age (day) 0.15 RORgt: WT S182A WT S182A ✱

γ 1500

p-S182 0.10 1000 Total RORgt 500 0.05

gMFI of ROR β-Tubulin

0 Normalized to Gapdh 0.00 β β Il17a

IL-6+IL-1 IL-6+IL-23 F. IL-6+IL-23+IL-1 Th17: ChIPseq (GSE40918) G.

RORgt IL-1b p300 IL-1 receptor Regions: CNS2 Il17a_P CNS3a CNS3b CNS4 CNS5 Il17f_P ChIP-H3K27ac ChIP-RORγt ChIP: RORgt ChIP: H3K27ac 0.6 15 RORγtWT S182 S182A p300 0.4 RORγt

10 t ✱✱ g ROR 0.2 5

% of the input % of the input H3K27ac CNS4 Il17a-f 0.0 0

Il17a_P Il17f_P Il17a_P Il17f_P Il17_CNS2 Il17_CNS4Il17_CNS5 Il17_CNS2 Il17_CNS4Il17_CNS5 Il17_CNS3aIl17_CNS3b Il17_CNS3aIl17_CNS3b (Neg) Il23r_int8 (Neg) Il23r_int8 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig 4. RORgtS182A T cells drived exacerbated diseases in two models of colitis.

A. B. RORγtWT Rag1-/- WT RORgt CD4+ RORγtS203A Il1r1Il1r1 Il23r Il17aIl17a T RORgtS182A ✱ 0.02 0.002 110 ✱✱ 0.003 ✱✱✱✱✱✱ ✱ 100 0.002

90 0.01 0.001 0.001 80

Normalized mRNA

Normalized mRNA Normalized mRNA C. % of original weight 70 0.00 0.000 0.000 WT WT WT 0 2 5 10 13 16 19 21 24 27 29 31 t t t γ S182A γ S182A S182A t t γ γ γ γt Days after transfer ROR ROR ROR ROR ROR ROR

C. D. E. RORgtWT RORgtS182A

RORgtS182A

2% DSS H 2O RORgtWT Days: 0 7 10

S203A_DSS5.25.18 and 7.12.18 Colonic length_maleInflammatory mice Infiltrate

110 10 ✱✱ 4 ✱ ✱ ✱ ✱ 8 100 3 6 90 2

4 Score:

80 1 2

% original weight

Colonic Length (cm)

inflammatory infiltrate inflammatory 70 0 0

0 2 3 4 6 8 9 10 WTWT KI / KI WT t gt g S182A S182A t Days post 2% DSS gt g ROR ROR WT (n=5) ROR ROR

S203A_KI/KI (n=6) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig 5. scRNA-seq revealed RORgtS182-dependent colonic Th17 and Treg programs during DSS challenge.

A. B. C. ROR�t+ ROR�t+ ROR�t- Foxp3- Foxp3+ Foxp3+ Cluster: 0 2 3 0 2 3 Cluster: 0 2 3

DSS: − − − + + + WT Rorc t � ROR Il17a S182A t Il1r1 g / WT) 17A - ROR IL S182A

t IL-10 g Ccr7 S203A weight + + 60 IL-17A 80 IL-10 ✱ 40 ✱✱✱

Expression Level Expression WT WT S1pr4 + RORgt 60

40 + RORRORgtγS182AtS203A 30 40 % fold change (ROR change fold

2 Foxp3 20 % of IL-10 % of IL-17A 20 Log 20

Weight (g) 0 ROR�tWT + + − − + + − − + + − − S182A - - 10 RORgt − − + + − − + + − − + + + + - + + - DSS − + − + − + − + − + − + + Foxp3 - - Foxp3 - + Foxp3 Foxp3 + Foxp3 + Foxp3 - Foxp3 Foxp3 t t t γt t t t γ γ γ γ γt γ γ 0 ROR ROR ROR ROR ROR ROR ROR ROR 54 60 64 68 70 73 78 85 91 98 109 114 119 131 141 150

0 Suppressive Cluster 2 1 NLT Age (day) D. 2 LT-like subsets 3 LT-like E.

4 Suppressive 2.00 Il10 Ctla4 Ccr5 Maf Fgl2 Icos Il4ra Ccr2 Itga6 Cd44 Tgfb1 Gzmb Ccr1 Areg Cmtm7 Lgals1 Il1r2 Cxcl3 S100a4 Ccr7 Sell S1pr1 Cxcr6 Furin Hif1a Itgal Bcl2 Tcf7 Tnfsf8 Pim1 Relb Ccl5 Stat4 Il18r1 Itgav Tnfrsf4 Tnfrsf9 Itgb8 Nr4a1 Cd83 Rora Pdcd1 Klrg1 Ikzf2 Il1rl1 Gata3 Stat1 Kdm6b Nr4a3 Itgae

1RNA.1

3RNA.3

2RNA.2 0.00 4RNA.4

0RNA.0 id Cluster 2 subsets Mean scaled exp -2.00 0.00 2.00

id RNA.0 RNA.4 RNA.2 RNA.3 RNA.1 id Il10 F. -2.00 G. Ctla4 Ccr5 DSS: Cluster 2 subset 0 DSS: Cluster 2 subset 3 All cells: MafCluster 2 subset 3: Fgl2 Icos Il10 Il4ra Il10 Ccr2 Itga6 Cd44 Tgfb1 Gzmb Ccr1 Areg cells % cells Cmtm7 Lgals1 Cluster 2 Il1r2 Cxcl3

S182A S100a4

t Ccr7

g Sell S1pr1 Cxcr6 Furin Hif1a ROR WTItgal RORgt Bcl2 + + − − S182ATcf7 RORgt Tnfsf8 − − + + WT WT Pim1 RORgt cells % RORgt cells % DSS Relb − + − + Ccl5 Stat4 Il18r1 Itgav Tnfrsf4 Tnfrsf9 Itgb8 Nr4a1 Cd83 Rora Pdcd1 Klrg1 Ikzf2 Il1rl1 Gata3 Stat1 Kdm6b Nr4a3 Itgae bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

g S182A Fig 6. S203AROR weightt mice experienced more severe disease in EAE model. 40 RORWT gtWT A. B. C. WT (n=7) RORRORgtγS182AtS203A 30 EAE Spinal Cord: Th17 RORγtS203A (n=14)

6 120 pRORgt ** *** 20 *** 4 110 * ****** Weight (g) IL-17A 10 Recruit & stimulate 2 100

Disease score 0 0 Known % of original weight Tgd17 Recruit CD11c 54 60 64 68 70 73 78 85 91 98 -2 80 IL-17A more T 109IL114-1β119 131 141 150 eff 1 9 12 14 16 19 21 23 26 28 1 9 12 14 16 19 21 23 26 28 Age (day) Days after immunization Days after immunization

D. E. EAE-peak

0.03 100 ✱ ✱ 10-1 ✱ 0.02 10-2

10-3 0.01 10-4 Normalized mRNA Normalized mRNA

0.00 10-5

Il6 Cd4 Ifng Il1b Il10 Il13 Il22 Cd19 Csf2 Il17a Il17f Il23a Tgfb Cd11c Cd11b Il12p40 F. G.

0.8 0.08 0.25 0.02 ✱ ✱ ✱ ns WT WT 0.20 0.6 0.06 S203A_KI/KI S203A_KI/KI 0.15 0.01 0.4 0.04 0.10 ✱✱ ✱ 0.2 0.02 0.05 Cell number (x10^6) Cell number (x10^6) Cell number (x10^6) Cell number (x10^6) 0.0 0.00 0.00 0.00 + - - + 17 γδ γδ + Th17 γδ + F4/80 + CD3e + T + TCR - TCR

IL-17A CD11b CD11c CD4 CD4 IL-17A bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig S1. Normal thymic T cell development and intestine homeostasis in RORgtS182A mice.

40 A. B. WT S203A 30 RORγt Human RORgt: SCHLEYSPERGKAE RORgtWT Mouse RORgt: SCHLEYSPERGKAE 20 RORgtS182A Weight (g) 10 AF-1 DBD LBD

0 0 50 100 150 200 Age (day)

C. Thymus E. Thymus F. Spleen 4000

3000

S203A weight t of DP γ WT t WT g 2000 t

40 g RORWT gtWT ROR 1000 RORRORgtγS182AtS203A ROR 30 GMFI of ROR 0

20 D. WT KI/KI S182A t Weight (g) S182A g t g 10 Max ROR ROR CD4 0 CD4 CD8a CD8a Bcl-xL 54 60 64 68 70 73 78 85 91 98 109 114 119 131 141 150 Age (day) G. H. Small intestine Small intestine Colon Colon 40 2.0 40 8 RORγtWT S182A 6 30 1.5

RORγt - - 30 + + + + 20 1.0 20 4 % % of TF % of % of CD4 % of CD3 of % CD3 of % % of CD4 of % CD4 of %

% of CD4 10 0.5 10 2

0 0.0 0 0 - ILC3 + + ILC3 + - + + RORgt+ RORgt (ILC3) (ILC3) + Foxp3 + Foxp3 - Foxp3 t t t + Foxp3 + Foxp3 - Foxp3 γ γ γ t t t γ γ γ ROR ROR ROR ROR ROR ROR cells cultured from WT and and WT from cells cultured Fig S2. A. bioRxiv preprint Representative cytokine production potential inTh17 was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. RORgtS182A RORgtWT doi:

IL-17A https://doi.org/10.1101/2021.05.14.444071 IL - 17F - TGFβ ROR ; IL this versionpostedMay16,2021. IL g - - t 1β 6 S182A mice IL - 23 . The copyrightholderforthispreprint(which IL - 1β+IL - 23 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig S3. Cell surface IL-1R expression on colonic Th17 cells in steady state and DSS-challenged WT and RORγtS182A mice.

DSS: − +

Gate: RORγt+Foxp3- WT t g 40 ✱ ROR 30 +

20 S182A t g

% of IL-1R 10 ROR 1R 0 - IL DSS:Steady − DSS + FSC bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig S4. DSS-induced epithelial injury altered colonic immune cell populations and their transcription programs in WT mice.

A. B. scRNAseq average exp

Clusters umap DSS umap Apoe Cd74 GenotypeCst3 umapIl1b 40 0 DSS 0 RORgt 1 No DSS WT 2 1 5 5 3 5 2% DSS H2O 4 5 2 6 30 Cluster 4 7 8 3 0 0 9 0 Cluster 10 Days: 0 7 10 10 4 11 5 20 UMAP_2 UMAP_2 UMAP_2 5 5 5 Il1b − − − 6 Clusters 7 10 10 10 10 8 − − − 9 10 15 15 15 − − − 0 −5 0 5 10 15 20 −5 0 5 10 15 20 11 −5 0 5 10 15 20 UMAP_1 UMAP_1 UMAP_1 DSS:Steady − DSS +

C. D. E.

6 Down in DSS Log2 fold change: DSS/Control5 (WT) Up in DSS 3 6 2 WT:6 DSS/No DownCluster: in 0DSS 3 2 1 5 Cluster 5 1 5 Up in DSS TNF signaling NF-kappa B signaling 3 0 3 ) Oxidative phosphorylation Val

p NOD-like receptor signaling 2 2 PI3K-AKT signaling 400 200 0 200 400Cluster Clusters (adj Antigen processing and presentation 1 1 10 # of DE genes Apoptosis Log 0 0 - RNA degradation RNA transport 400 200 0 200 400 -4 -2 0 2 4 # of DE genes bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig S5. RORgtS182-dependent cell populations and target genes in colon under steady state and during DSS challenge. A. DSS treated colon scRNAseq

40 DSS: WT vs KI DSS_WT ✱ 30 30 ✱ DSS_RORDSS_KIγtWT 20 DSS_RORγtS182A 20 % of total 10

10 0 0 1 2 3 5 6

% of lamina propria cells Clusters 0 0 1 2 3 4 5 6 7 8 9 10 11 B. Clusters

Steday state DSS

S182A S182A 6 6 RORγt _down RORγt _down S182A S182A 5 5 RORγt _up RORγt _up

3 3

2 2 Cluster Cluster 1 1

0 0

200 -100 0 100 200 -100 -50 0 50 100 # of DE genes # of DE genes bioRxiv preprint doi: https://doi.org/10.1101/2021.05.14.444071; this version posted May 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig S6. Cluster 2 Treg subsets in control and DSS- challenged WT and RORgtS182A mice.

Steady state colon scRNAseq

80 Control_WT 60 RORgControl_KItWT

40 ✱ RORgtS182A

20 % of Cluster 2

0 0 1 2 3 4 sub-cluster

DSS treated colon scRNAseq

40 DSS_WT 30 RORgtDSS_KIWT

S182A 20 RORgt

10 % of Cluster 2

0 0 1 2 3 4 sub-cluster