DR.

SONG ZHANG (Orcid ID : 0000-0003-1492-8988)

Accepted Article
Article type : Review

The role of TGF- in Th17 differentiation

Song Zhang1,2

1
State Key Laboratory of Medicinal Chemical Biology; College of Life Sciences, Nankai

University, Tianjin 300071, China.

2
Lineberger Comprehensive Cancer Center; Department of Microbiology and Immunology,

University of North Carolina at Chapel Hill, North Carolina 27599, USA.

*Correspondence: sz@unc.edu

Summary:

Th17 cells play critical roles in inflammatory and autoimmune diseases. The lineage-

specific transcription factor RORt is the key regulator for Th17 cell fate commitment. A

substantial number of studies have established the importance of TGF--dependent

pathways in inducing RORt expression and Th17 differentiation. TGF- superfamily

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/imm.12938

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 members TGF-1, TGF-3, or activin A, in concert with IL-6 or IL-21 differentiate naïve T

cells into Th17 cells. Alternatively, Th17 differentiation can occur through TGF--
Accepted Article
independent pathways. However, the mechanism of how TGF--dependent and TGF--

independent pathways control Th17 differentiation remains controversial. This review

focuses on the perplexing role of TGF- in Th17 differentiation, depicts the requirement of

TGF- for Th17 development, and underscores the multiple mechanisms underlying TGF-

promoted Th17 generation, pathogenicity and plasticity. With new insights and

comprehension from recent findings, this review specifically tackles the involvement of the

canonical TGF- signaling components, SMAD2, SMAD3 and SMAD4, summarizes diverse

SMAD-independent mechanisms, and highlights the importance of TGF- signaling in

balancing the reciprocal conversion of Th17 and Treg cells. Finally, this review includes

discussions and perspectives and raises important mechanistic questions about the role of

TGF- in Th17 generation and function.

Keywords: Th17 differentiation, TGF-, SMAD

Cytokine milieu for Th17 differentiation

T helper 17 (Th17) cells were identified as a new subset of CD4+ T helper cells

producing interleukin-17 (IL-17) in 20051, 2. In response to environmental cues, naïve T cells

differentiate into Th17 effector cells (Fig.1). Th17 differentiation requires both inflammatory

cytokine (e.g., IL-6, or IL-21) and TGF- to efficiently induce the lineage-specific transcription

factor RORt as well as IL-17 production3. Th17 cells are critically involved in host defense,

inflammation and autoimmunity4. Th17 responses contribute to the pathogenesis of diverse

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 human diseases, including psoriasis, asthma, rheumatoid arthritis, inflammatory bowel

disease and multiple sclerosis5, 6. A better understanding of Th17 differentiation therefore

Accepted Article
would lay the basis for therapeutic interventions7.

Inflammatory cytokines

IL-6 is characterized as an inflammatory cytokine together with TGF- featuring Th17

induction activity8, 9. Engagement of IL-6 with its receptor leads to activation of STAT3, which

further potentiates RORt transcription4. Although various hematopoietic and non-

hematopoietic cells can produce IL-6, a recent study showed that Sirp+ dendritic cells

(DCs) are especially essential for pathogenic Th17 cells by trans-presenting IL-6 to T cells

during the process of cognate interaction10.

As an alternative cytokine, IL-21 also can stimulate STAT3 activation. IL-21 in

combination with TGF- leads to RORt expression and Th17 differentiation independent of

IL-611-14. In addition, IL-6 induces IL-21 during Th17 differentiation to further enhance the

Th17 program.

Other cytokines also play important roles in the development of Th17 cells. IL-1

signaling is indispensable for Th17-mediated autoimmunity in experimental autoimmune

encephalomyelitis (EAE)15, and is necessary for early Th17 differentiation in vivo16. It is also

critical for the expansion, but not the generation, of autoreactive GM-CSF+ Th17 cells17.

Additionally, IL-1 enhances Th17 differentiation by inhibiting TGF--induced Foxp3

expression18.

IL-23 is capable of activating the STAT3 pathway and promoting Th17 lineage

maintenance but not commitment19. Importantly, T cells exposed to IL-23 have promoted

expression of GM-CSF and IL-23R to become more pathogenic20, 21.

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 Thus far it is still unclear if there are other unrecognized cytokines that can act

functionally like IL-6 or IL-21 to initiate Th17 differentiation with TGF-. It has also been
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elusive whether STAT3 activation is the only major function for IL-6 and IL-21 to induce

Th17, or whether these cytokines have other important targets to synergize with TGF-

signaling and induce Th17 differentiation.

Requirement of TGF- for Th17 differentiation

TGF- is a regulatory cytokine, exerting pleiotropic functions in T cell development,

homeostasis, tolerance, and differentiation22, 23. TGF- is produced as an inactive form in

complex with latency-associated peptide (LAP) and latent TGF--binding protein (LTBP),

and is further activated by disengaging from this large latent complex (LLC). Multiple

mechanisms are involved during this process, including plasmin, MMPs, TSP-1, and

integrins22, 24, 25
. Integrin binds to LAP and induces the release of mature TGF-, which

provides an additional layer of mechanistic regulation. For example, although dendritic cells

do not produce TGF-, these cells are important for v8 integrin-mediated TGF- activation

to exert biological functions such as inducing Th17 differentiation26. The v8 integrin

transcription in APCs depends on IRF8. Mice with IRF8 deficiency in APCs, but not T cells,

fail to generate Th17 cells and were resistant to EAE27.

TGF- established its requirement in murine models shortly after Th17 cells were

identified8, 9, 28. TGF-1 deficient mice exhibited profoundly diminished Th17 cells in all tissue

sites27. Mice with TGF-1 deficiency in T cells were also defective in Th17 differentiation as

well as the onset of EAE29. Mice with TGF- signaling blockade by a dominant negative form

of TGF- receptor II (CD4dnTGFRII) or by a TGF--specific antibody failed to differentiate

naïve T cells to Th17 cells and were protected from EAE30, while TGF- transgenic mice

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 resulted in enhanced Th17 differentiation and more severe EAE8. These data strongly

suggest TGF- is indispensable for Th17 differentiation.

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Initially, human cells were considered not to require TGF- but only IL-6 and IL-1, or

IL-1 and IL-23 for Th17 differentiation31, 32. Naïve CD4+ T cells (defined by CD4+CD45RA+

CD45RO- 32
, or CD4+CD45RA+CCR7+CD25- 31
) used in these studies were sorted from

peripheral blood, and thus raised the concern of naiveté33. In addition, there was possible

TGF- contamination from the serum of culture medium. Therefore in later studies, naïve

cord blood CD4+ T cells (defined by CD3+CD4+CD25−HLA-DR−CD45RA+ 34

,

CD3+CD4+CD45RA+CD45RO− 35
, or CD4+CD25-CD62L+CD45RAhi 36
) and serum-free

medium were used. With minimized TGF- source contamination from serum or platelets,

and cord blood originated naïve CD4+ T cells, these studies clarified that TGF- is indeed

required for human cell Th17 differentiation34-36. CD161+CD4+ T cell precursors in umbilical

cord blood and thymus were reported to constitutively express RORt, IL-23R, and CCR637,
38
. These cells produced IL-17 in response to IL-1 and IL-23 without the need for TGF-37, 39.

Because most of the CD161+ cells express CD45RO37, 40, these precursor cells might have a

memory T cell phenotype. IL-1 and IL-23 could contribute to cell activation or expansion

rather than de novo Th17 differentiation. Moreover, TGF- is potent for skewing these

CD161+ cells from Th1 towards Th17 after IL-1 and IL-23 stimulation39. Collectively, these

data suggest that TGF- plays an essential role in human Th17 differentiation.

TGF- source, TGF- superfamily, and Th17 cell pathogenicity

There are three isoforms of TGF-: TGF-1, TGF-2, and TGF-341. Vast types of

cell produce TGF-1, and T cells are the important source of TGF-1 to promote Th17

differentiation and to develop EAE29, although the role of TGF-1 in EAE induction is

controversial42-44. Despite the fact that Th1, Th2, and Th17 cells can express TGF-1 after

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 polarization, Th17 effector cells have the most increased production of TGF-1. In vivo Th17

differentiation requires the autocrine TGF-145. However, this finding is not consistent with
Accepted Article
the observation that autocrine TGF- produced by in vitro differentiated Th17 cells under IL-

6+IL-1+IL-23 condition is not essential, as TGF- antibody blockade does not significantly

reduce Th17 differentiation46. Therefore, further debate on the role of autocrine TGF-

produced by Th17 cells continues.

Foxp3+Treg cells could serve as another source of TGF-1 production, which may

induce naïve T cell to differentiate into Th17 cells under in vitro co-culture conditions9.

However, mice with TGF-1 deletion specifically in Foxp3+Treg cells result in similar level of

Th17 differentiation during an in vivo model of EAE, arguing that Foxp3+Treg derived TGF-

1 is not essential for Th17 differentiation45.

TGF-1, produced by Treg cells or activated CD4+ T cells, does not substantially

affect the T cell activation in spleen and lymph nodes, but it is required for inhibiting Th1

differentiation in the gut45. A subset of Tregs, CD103+Treg cells, possess very high

suppressive activity and exist at sites of inflammation and mucosal tissues including gut47-49,

suggesting that the different microenvironment and local cytokine milieu may determine how

TGF- influences Th17 differentiation and propagates disease progression50.

The sources of TGF- include stromal cells, immune cells and cancer cells, which

provide a basis for versatile regulation in local immune responses23. For example, gliadin-

specific Th17 cells from celiac disease patients simultaneously express TGF-. This

autocrine TGF- plays a positive regulatory role in IL-17 production in intestinal mucosa51.

TGF- prevails in the intestine, and intestinal epithelial cells (IECs) and DCs are important

sources of bioactive TGF-. In addition, CD103+DCs express a high level of integrin v8,

and are specialized to activate latent TGF-52-54. In healthy humans, about 4ng/ml TGF-1

circulates in plasma, therefore it may act in a manner of long-range endocrine interactions

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 between distant tissues. However, TGF-2 and TGF-3 are likely derived from local

synthesis, as they are generally undetectable in plasma, and appear to act as local autocrine
Accepted Article
or paracrine regulators.55

TGF- not only promotes Th17 differentiation but also determines the pathogenicity

of Th17 cells. Researchers observed that TGF-1-induced Th17 cells are less pathogenic in

EAE20, while TGF-3 leads to more pathogenic Th17 cells21. TRIM28 deficient T cells cause

aberrant overexpression of TGF-3 and contribute to the development and accumulation of

pathogenic Th17 cells56.

Other than TGF- family cytokines, a TGF- superfamily member, activin A, was also

reported to be capable of inducing Th17 differentiation in combination with IL-657, 58.

Because there are more than 33 human TGF- superfamily members, including

TGF-, bone morphogenetic proteins (BMPs) and activins, and they share similar

structures23, it would be important to identify if there are more cytokines that could be

functionally similar to TGF-1, TGF-3, and activin A during Th17 differentiation.

The diverse functions of the TGF- superfamily rely on their specific receptor

signaling, which goes through different heteromeric type I and type II receptor complexes.

Receptors TGFRI (ALK5) and TGFRII are mostly designated to TGF-, whereas receptors

ACTRIB (ALK4) and ACTRIIs receive signaling from activins23, 59

. The differences in

receptors could explain why activin A alone fail to induce Foxp3 while TGF- can induce

Foxp360. However, TGF-1 and TGF-3 share identical receptors, yet lead to distinct

pathogenic profiles in Th17 cells21. This suggests that there might be other undefined

receptors or mechanisms in existence, which requires further investigation.

Th17 cells are recognized as important pathogenic effector cells in most EAE

models61. EAE is attenuated when mice are injected with anti-IL-17A antibody2, 62, 63 and IL-

17RA deficient mice are resistant to EAE64. IL-17A deficiency results in reduced clinical EAE,

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 although it does not fundamentally impede the incidence of disease induction65, 66. Cytokines

critical for Th17 differentiation, such as IL-667, 68

, IL-2111, 12
, IL-115, and IL-2369, are also
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required for EAE. Increasing evidence shows that several Th17 expressed genes are

especially vital for the pathogenicity of Th17 cells. For example, IL-23R expression is shown

to be critical for the generation of encephalitogenic Th17 cells70, 71, and GM-CSF deficient

mice are resistant to EAE72-75. It appears that there are certain molecular networks

associated with the pathogenicity of Th17 cells. Different TGF- signaling pathways produce

different pathogenic programs21, 46, 76

. As the Th17 cells are hightly heterogeneous, the

diversity of TGF- superfamily ligands and receptors provides a tool for investigating the

essential mechanisms of Th17 pathogenicity.

TGF--independent Th17 cell differentiation

There are also studies showing that TGF- is dispensable for murine Th17

differentiation. In the presence of anti-TGF- antibodies, STAT6 and T-bet double deficient T

cells still can differentiate into Th17 cells with IL-6 alone77, 78. These observations raise the

debate on the requirement of TGF- in Th17 differentiation. However, TGF- antibody

blockade, but not TGF- receptor signaling deficiency, could not rule out the possibility that

there is still TGF- or that TGF- superfamily receptor signaling exists in this settings. Later,

Ghoreschi et al. reported that although TGF- receptor I deficient mice or CD4dnTGFRII

mice were not able to induce EAE, there were similar amounts of Th17 cells in intestinal

lamina propria as compared to wild type mice46. They also found that Th17 cells could be

generated in vitro without TGF- using a combination of cytokines (IL-6, IL-1, and IL-23)

and that these Th17 cells were more pathogenic. These data suggest an alternative TGF--

independent Th17 differentiation pattern. It also provides insight and opportunity to

investigate the mechanism of TGF--dependent and TGF--independent Th17 differentiation

and Th17 cell pathogenicity. Compared to TGF-, this cytokine combination had a relatively

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 low ability to induce in vitro Th17 differentiation16, 46, but enough to raise the argument that

TGF- may not be necessary under certain environmental context.

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To date, the mechanisms of how these Th17 cells are induced by cytokine

combinations without requiring TGF- signaling, and how the downstream receptor signaling

of IL-6, IL-1 and IL-23 synergized, are still perplexing. Notably, TGFRI ablation, but not

TGF- antibody blockade, dramatically reduced the proportion of Th17 cells from 30 percent

to 13 percent46. This suggests that TGFRI signaling at least partially contributes to Th17

differentiation under this cytokine regimen. It is very likely that there is TGFRI ligand

produced, which enhances the Th17 program in an autocrine pattern under these in vitro

culture conditions. Further studies should especially focus on TGF- superfamily cytokines

that can be secreted by these Th17 cells and also can signal through TGFRI. For those

Th17 cells deficient in TGFRI, it is also possible that they are primed by the combination of

cytokines, and then secondarily induced by autocrine cytokines through other TGF-

superfamily receptors except TGFRI. Further studies are warranted to uncover the

requirements and central mechanisms of TGF- signaling in Th17 differentiation.

Mechanisms underlying TGF- promoted Th17

differentiation

TGF- induces naïve T cells to express the regulatory transcription factor Foxp3

upon TCR activation79. Foxp3 can directly interact with RORt and antagonize the Th17

master regulator RORt80-82. Although IL-6 can activate STAT3 and inhibit Foxp3 expression8,

it cannot sufficiently induce the RORt transcriptional program. TGF- is required for the full

differentiation capacity of Th17 cells. Confounded by these observations, extensive studies

are performed to explain the paradoxical role of TGF- in Th17 differentiation in the last

decade (Fig. 2).

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 Essential transcription factors in Th17 differentiation

Th17 differentiation is controlled by several key transcription factors83. Th17 lineage-

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specific regulator RORt (encoded by Rorc gene) directs IL-17 production by binding to the

IL-17 gene locus3, 81

. RORt deficient T cells fail to become Th17 cells. Reducing the

transcriptional activity of RORt by antagonist such as digoxin effectively inhibits Th17

differentiation84. Posttranslational regulation of RORt by small-molecule RORt

antagonists85, or by ubiquitin ligase UBR5 in response to TGF- signaling can limit the

abundance of RORt and thus prevent rampant IL-17 production86.

STAT3 regulates the transcription of its multiple target genes, including Rorc, Il17a,

and Il17f. STAT3 is required for Th17 differentiation. STAT3 deficiency results in abrogated

Th17 generation, while overexpression of STAT3 significantly boosts Th17 differentiation14,

87-89
.

The cooperative binding of BATF and IRF4 is critical for initial chromatin accessibility

and combinatorial regulation with STAT3 during Th17 differentiation83, 90

. Either IRF4 or

BATF ablation results in failure of RORt induction and defective Th17 differentiation91-93.

SMAD-dependent pathways

The canonical TGF- pathway signals through the TGF- receptor and

phosphorylates receptor-regulated SMADs (R-SMAD), SMAD2 and SMAD3. The

heterodimeric complex then interacts with the common mediator SMAD (co-SMAD), SMAD4,

to generate a heterotrimeric complex. This new complex translocates into the nucleus for

downstream gene modulation. Complementarily, TGF- can activate non-canonical

pathways such as PI3K/AKT/mTOR, MAPK pathways (ERK, JNK, and p38 MAPK), and NF-

B pathways for T cell function94-96.

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 SMAD2 deficient T cells have reduced ability for Th17 differentiation57, 97
. SMAD2

may modulate IL-6R expression and STAT3 activation in the presence of TGF-39. Another
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study found that SMAD2 binds to and synergizes with RORt and positively regulates Th17

differentiation57.

SMAD3 appears to act in opposition to SMAD2. Deficiency of SMAD3 leads to

enhanced Th17 differentiation both in vitro and in vivo98. SMAD3 is also found to bind

directly to RORt, and inhibits RORt transcriptional activity.

There is also an argument that neither SMAD2 nor SMAD3 is essential for Th17

differentiation. In one report, no defect in Th17 differentiation in either SMAD2 or SMAD3

deficient mice was observed, whereas the non-canonical MAPK pathway was implicated99.

In another report, Th17 differentiation in SMAD2 deficient mice was reduced, but remained

intact in SMAD3 deficient mice, and was abolished in SMAD2 and SMAD3 double knockout

mice100. The deficiency of SMAD2 and SMAD3 did not alter Rorc gene expression but

increased the level of the inhibitory cytokine, IL-2101. In the presence of IL-2 blocking

antibody, CD4+ T cells carrying SMAD2/3 double knockout restored half of the efficiency to

differentiate into Th17 cells. These results suggest SMAD2 and SMAD3 are partially and

redundantly required for Th17 differentiation. A recent report supports the idea that SMAD2

plays a positive role, while SMAD3 plays a negative role in regulating Th17 differentiation102.

SMAD2 serves as a co-activator, and SMAD3 serves as a co-repressor in interactions with

STAT3 to modulate Rorc and Il17a gene expression under the context of TGF- determined

phosphorylation status.

These conflicting results regarding SMAD2 or SMAD3 deficient mice among different

studies appear confusing. This could be due to differences between wild type control cells

and knockout cells that were compromised under certain in vitro culture conditions or

treatments, including TGF- dose. Different doses of TGF- result in different R-SMAD

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 phosphorylation dynamics103, and different efficiencies in Th17 induction4. Nonetheless, all

these studies acknowledge the involvement of the canonical TGF- pathway in Th17
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differentiation.

SMAD4 is recognized as a canonical TGF- signaling central component. So it is

reasonable to predict a TGF- signaling defect after deletion of SMAD4. Indeed, TGF--

induced Foxp3+Treg differentiation is impaired in SMAD4 deficient mice. Astonishingly,

however, Th17 differentiation is unaffected by the loss of SMAD4 under Th17 polarizing

condition104. This observation is confirmed later by another study105. Therefore, it appears

that SMAD4 is not required for Th17 differentiation, and TGF- induces Th17 differentiation

through a SMAD4-independent mechanism. However, in later studies, SMAD4 deletion

counterbalances the severe autoimmunity caused by the loss of TGF- receptor II signaling,

suggesting that SMAD4 could regulate T cell function on its own in the absence of TGF-

receptor signaling106. Inspired by the notion, Zhang et al. fortuitously found that SMAD4

deficient naïve T cells efficiently differentiated into Th17 cells when provided with IL-6 alone

in the absence of TGF- signaling58. Although TGF- receptor II mice were unable to

generate Th17 cells in the spinal cord and were protected from EAE, the additional deletion

of SMAD4 fully restored Th17 differentiation as well as encephalomyelitis. These data

suggest that SMAD4 is playing a decisive role in licensing Th17 differentiation. A series of

experiments demonstrate that SMAD4 and repressor SKI interact and cooperatively

suppress Th17 differentiation primarily through direct binding to multiple loci of Rorc. SMAD4

itself does not possess the suppressive activity but it is required for recruiting SKI to specific

chromatin loci. The suppression effect can be offset in the presence of TGF- through TGF-

 directed degradation of SKI. These observations provide novel mechanistic insight into

TGF--dependent Th17 differentiation. CD4+ T cells with CRISPR/Cas9 disrupted SKI can

differentiate into Th17 cells without the need for TGF- signaling. This suggests one

important function for TGF- in Th17 differentiation is to degrade SKI. Further studies on SKI

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 conditional knockout mice would be helpful to elaborate on the mechanistic details.

Importantly, a single amino acid substitution, which released SKI from SMAD4 interactions
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caused unchecked Th17 differentiation, mutually affirms the importance of losing SKI

interactions in generating Th17 cells. Also, Ong et al. found that the absence of SKI in CD4+

T cells led to increased Th17 differentiation at low levels of TGF-107. Indeed, low doses of

TGF- can effectively degrade SKI and the degradation efficiency correlated with the

induction efficiency of Th17 differentiation58. The SKI/SMAD4 complex is highlighted as a

switch for Th17 differentiation, and it might be also helpful to explain the generation of

pathogenic Th17 cells by TGF--independent cytokines46.

Many questions remain for further mechanistic details, such as: why SMAD4 and SKI

primarily suppress the Rorc gene but not other genes? Is it possible that SMAD4 affects the

genome accessibility on the Rorc locus? Is the binding on the Rorc locus constitutive or

dependent upon specific TCR or cytokine stimulation? Since SKI could serve as a potential

therapeutic target for many diseases108, it would be important to investigate how SKI is

degraded by TGF- signaling in T cells. SMAD2 and SMAD3 were reported to mediate the

ubiquitination and degradation of SKI109, which may explain their partial requirement in T

cells58. However, TGF- signaling does not always trigger SKI degradation in all cell types110.

These questions must be addressed in future investigations.

SMAD-independent pathways

In addition to SMADs, there are several studies that offer insights from non-SMAD

pathways to explain the role of TGF- in promoting Th17 differentiation.

It has been well known that both Th1 and Th2 cytokines potently inhibit Th17

differentiation1, 2. TGF- is capable of inhibiting Th1 and Th2 differentiation through their

lineage transcription factors T-bet and GATA329, 111, 112

. TGF- may exert its function by

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 preventing Th1 and Th2 programs to enhance Th17 differentiation. In T-bet and STAT6

double deficient T cells, IL-6 alone is sufficient to induce Th17 differentiation77, 78

. These
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results clearly showed that one of the mechanisms for TGF- in promoting Th17

differentiation is to negatively regulate Th1 and Th2 programs. Currently, it is not clear

whether the double deficiency of T-bet and STAT6 can produce similar results in C57BL/6

mice as in these BALB/c mice. Notably, in these double knockout T cells, the addition of

either TGF- or anti-TGF- antibody had no effect on IL-17 production in one report78, while

it had around 4 times promotion or reduction effect on IL-17 production in another report77.

This discrepancy and genetic background difference obscure the interpretation of how

exactly TGF- plays the role in Th17 differentiation. As SKI/SMAD4 also functions as a

suppressor of RORt expression, and SMAD4 deficient T cells also can differentiate into

Th17 cells with IL-6 alone, it is not clear whether these two mechanisms are required for

each other. For example, whether T-bet and STAT6 double deficient T cells have disrupted

SMAD4 or SKI function. STAT6 downstream target GATA3 was also reported to control

TGF--induced Foxp3, suggesting possible cross talk between Th1/Th2 and the downstream

of TGF- pathways113, 114.

IL-6-induced SOCS3 is a negative feedback regulator of STAT3, and TGF- can

suppress the transcription of SOCS3115. siRNA knockdown of SOCS3 promotes Rorc

expression and in vitro Th17 differentiation in the presence or absence of TGF-. These data

indicate that TGF- suppresses SOCS3 to prolong STAT3 activation and to promote Th17

generation.

Under Th17 skewing condition, protein kinase ROCK2 phosphorylated IRF4 to

upregulate the expression of RORt, IL-17, and IL-21. TGF- is required for driving the

activation of RhoA/ROCK2 to promote Th17 differentiation116.

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 In order to identify the SMAD-independent mechanisms underlying TGF- in Th17

cells, Ichiyama et al. utilized SMAD2 and SMAD3 double deficient mice and identified the
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transcription factor Eomesodermin (Eomes), which directly binds to the Rorc and Il17a

promoter region controlling Th17 differentiation117. Ablation of Eomes expression by shRNA

led to the induction of Th17 cells in the absence of TGF-, while overexpression of Eomes

substantially constrained Th17 differentiation117, 118. Eomes is subjected to the suppression

of TGF- signaling through JNK-c-Jun pathway, which provides an important mechanism for

SMAD-independent Th17 cell differentiation.

In summary, these SMAD-dependent and SMAD-independent pathways prove that

TGF- induces Th17 differentiation not through a single pathway but rather that multiple

mechanisms are involved to achieve efficient Th17 differentiation.

In recent years, there has been an increased understanding of environmental cues

shaping the Th17 differentiation119-121. For example, Serum glucocorticoid kinase 1 (SGK1)

can be upregulated by sensing the NaCl in a high salt diet through phosphorylation of p38

mitogen-activated protein kinase (MAPK), which promotes IL-23R expression, induces

pathogenic Th17 cells, and exacerbates murine EAE122, 123

. Since TGF-1 is able to

stimulate the SGK1 expression, and SGK1 is highly specifically expressed under Th17

conditions122, it could be another explanation for TGF--induced Th17 differentiation.

Although there have been arguments regarding the physiological underpinnings of this

model124-126, these data provide a novel target for drug development and methods for the

treatment of autoimmune diseases.

Although there are possibly more unrevealed TGF- signaling pathways for

promoting Th17 differentiation, further studies will help to elucidate the precise mechanism

of Th17 differentiation, as well as the pathogenicity of Th17 cells. Also, investigating the

prominent pathways under different pathological conditions and environmental cues would

be helpful for the intervention in Th17 differentiation to protect health.

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 TGF- signaling in Th17 cells plasticity

In addition to Th17 cells, naïve T cells can differentiate into several types of helper T
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cells (Fig. 3). Increasing evidence has implicated the plasticity of T cell subsets127. Since

TGF- is essential for the development of both Th17 and Foxp3+Treg cells, the TGF-

signaling pathway provides a dichotomous role and intimate link between Th17 and

Foxp3+Treg cells128, 129. TGF- can induce Foxp3 expression, while the presence of IL-6 or

IL-21 can prevent the conversion of Th17 to Foxp3+Treg cells and promotes Th17 lineage

commitment 8, 11, 12.

The reciprocal differentiation of Th17 and Foxp3+Treg cells are involved in multiple

immune mechanisms. TGF- signaling plays a critical role in the fate decision between Th17

and Foxp3+Treg cells. For example, TGF--induced Foxp3+Treg cells promote Th17

development through IL-2 regulation101, 130, 131. Mice deficient in TRAF6 have less IL-2 and

therefore have promoted Th17 cell differentiation132. In another study, TGF- down-regulates

the expression of a transcriptional repressor, GFI-1, which associated with histone lysine-

specific demethylase 1 to directly bind the intergenic region of Il17a/Il17f loci, thus represses

IL-17 expression49. Another example in which TGF-β regulates the Th17 and Foxp3+Treg

cell response is the TGF--induced TAZ. TAZ is a coactivator of RORt, decreases

acetylation of Foxp3 to regulate the reciprocal balance between Th17 and Foxp3+Treg

cells133. In addition, TGF--induced death-associated protein kinase (DAPK) promotes the

proteasomal degradation of HIF-1, a key metabolic sensor134, to regulate the balance of

Th17 and Foxp3+Treg cells, which ultimately prevents Th17-mediated pathology in

autoimmunity135. In another model, TGF- promoted colon-specific trafficking molecule

GPR15, which inhibits GPR174 on Foxp3+Treg cells, and supports the accumulation and

retention of Foxp3+Treg cells in the colon to dampen Th17 responses136. Finally, TGF-

triggers the degradation of the newly identified T cell player SKI, and promotes Th17

differentiation58. However, the deletion of SKI in Foxp3+ T cells also leads to increased

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 Foxp3+Treg conversion107. These observations suggest that residual amounts of SKI may

exist and play an important role in Foxp3+Treg cells, possibly balancing the differentiation of
Accepted Article
Th17 and Foxp3+Treg. Further study on the detailed role of SKI could be beneficial for the

understanding of Th17 and Foxp3+Treg reciprocal differentiation.

Th17 cells can also transdifferentiate into another regulatory T cell subset, type 1

regulatory T cell (Tr1) to halt inflammation137, 138. Besides, TGF- converts Th1 cells into

Th17 cells through the induction of RUNX1 expression139. Conversely, Th1 lineage-specific

transcription factor T-bet can repress RUNX1-mediated RORt transactivation to prevent

Th17 differentiation140. TGF- is required for human memory Th17 cells to produce Th9 cell

cytokine, IL-9141. Moreover, murine Th17 cells in Peyer's patches can convert to the T

follicular helper cells (Tfh) phenotype142, possibly because TGF- is critical for providing

additional signals for STAT3 and STAT4 to promote human Tfh differentiation143.

Currently, it is unclear if all T helper subsets have the potential to convert to each

other under specific circumstances. Although Th17, Foxp3+Treg, Tr1, Th9, and Tfh cells are

distinct subsets, all of these subsets can be promoted by TGF-138. It is possible that these

subsets share similar key factors associated with TGF- signaling pathway, and the factors

may be targeted as a switch in balancing autoimmunity and self-tolerance. It is thus

important to investigate the underlying mechanisms and identify the key factors in the TGF-

directed T helper programming network.

The importance of metabolic control in T cell function and plasticity has gained more

and more attention in recent years144-147. The mechanistic target of rapamycin (mTOR) forms

two structurally and functionally distinct complex, mTORC1 and mTORC2. mTOR senses

diverse environmental cues such as growth factors, oxygen, nutrients, energy, and stress,

and integrates into protein synthesis, lipid, nucleotide and glucose metabolism148. Loss of

mTORC1 results in failure of Th17 differentiation, while mTORC2 deficiency does not affect

Th1 or Th17 differentiation149, 150. Interestingly, if both mTORC1 and mTORC2 are inhibited,

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 T cells skew toward Foxp3+Treg differentiation. Although it remains unclear whether TGF-

plays a pivotal role in regulating the mTOR pathway under pathological conditions, the cross
Accepted Article
talk between TGF- and mTOR signaling is certainly worth of investigation. For example,

mTORC2 could modulate SMAD2/3 linker phosphorylation to regulate TGF- or activin

signaling151.

mTOR is required for the induction of hypoxia-inducible factor 1 (HIF1), a key

regulator mediating T cell glycolytic activity152. HIF1 controls the T cell fate decision

between Th17 and Foxp3+Treg. HIF1 deficiency alters the dichotomy between these two T

cell lineages by diminishing Th17 and promoting Foxp3+Treg differentiation134, 152. In vitro

experiments shows that TGF- has the ability to induce the expression of HIF1134, 153
.

However, the proportion and importance of TGF- contributed induction of HIF1 under in

vivo circumstance is currently unknown.

Myc is another important target of mTOR. Myc controls glycolysis and glutaminolysis.

It is required for metabolic reprogramming upon T cell activation154. The fact that Myc could

be repressed by TGF-155-157, suggests a possible link between TGF- and T cell metabolism.

Aryl hydrocarbon receptor (AHR) responds to environmental toxins and metabolites.

AHR boosts either Th17 or Foxp3+Treg differentiation in a ligand-specific fashion158, 159

.

Although AHR deficient T cells could differentiate into Th17 cells, they fail to produce the

Th17 cytokine, IL-22159. AHR is highly expressed in Th17 cells and Tr1 cells160, but not Treg

cells158, 159. AHR expression is positively correlated with TGF- concentration in the presence

of IL-6, but is not required for IL-22 production in the absence of TGF-161. TGF-

suppresses IL-22 production in Th17 cells through c-Maf but not AHR161. There are also

studies suggesting that the regulation of AHR by TGF- appears to be in a cell type specific

manner162-164. These paradoxes warrant further investigation on the role of TGF- in AHR

modulation.

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 These metabolic checkpoints and pathways orchestrate the balance between Th17

cells and other subsets165-167. The understanding of Th17 plasticity may provide tools for
Accepted Article
immunotherapies of autoimmune disease168, 169. However there are significant gaps between

the TGF- signaling and T cell metabolic control. Therefore, it is important to investigate how

TGF- shapes and affects metabolic reprogramming in T cells.

Concluding remarks and featured questions

The unravelling of the key factors and pathways responsible for both TGF--

dependent and TGF--independent Th17 differentiation is essential for understanding the

development, pathogenicity and plasticity of Th17 cells. With more understanding of the

mechanisms underlying TGF- promoted Th17 differentiation in the future, the following

questions should be addressed:

1. What are the dominant cytokine sources for tissue resident Th17 and pathogenic Th17

cells in different tissues or organs during health and disease conditions?

2. Is the dysregulation of TGF- or TGF- signaling responsible for the Th17 cells that have

gone rogue?

3. How is Th17 differentiation regulated by various cytokine regimens? Does the SKI/SMAD4

complex play a role in other cytokine signaling pathways besides IL-6 plus TGF-1?

4. Are there any other unknown key transcriptional suppressors or activators of RORt which

also respond to TGF-?

5. How exactly is the pathogenicity of Th17 cells regulated? What are the functions of TGF-

signaling in pathogenic Th17 cells? Does the TGF--SKI-SMAD4 axis play a role in the

generation of pathogenic Th17 cells?

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 6. Are there specific markers and functional differences to distinguish initially committed and

subsequently converted Th17 cells?

Accepted Article
7. Within the context of autoimmune disease, is it appropriate to always assume Treg cells

as friends while considering Th17 cells as foes? Do these cells work together during the

initiation or progression phase of the disease?

8. Do all T helper cell subsets have the plastic potential to become any other subset? Is it

possible to develop a drug to convert some, if not all, of the different subsets into one subset

simultaneously?

9. Is TGF- or TGF- signaling involved in the metabolic control of T cell function?

Further comprehensive studies on the role of TGF- in controlling Th17 differentiation

and function will allow us to gain insights on how Th17 differentiation impacts immune

homeostasis under physiological context and facilitates the development of therapeutic

intervention methods for inflammatory and autoimmune diseases.

Acknowledgements

The author thanks Seddon Y. Thomas for critical reading and editing of the manuscript. This

research was supported by State Key Laboratory of Medicinal Chemical Biology.

Disclosures

The author declares no conflict of interest.

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Figure legend:

Figure 1: Th17 differentiation

IL-6 or IL-21 activates STAT3 to potentiate Rorc expression, which encodes the RORt

transcription factor. IL-6 activates STAT3 through classic, trans or cluster signaling

pathways. Additional TGF- or activin A degrades SKI to reverse the SKI/SMAD4 complex

imposed repression on Rorc transcription. Unleashed RORt binds to the Il17a, Il17f loci and

drives T cells to produces Th17 cytokines, e.g., IL-17A, IL-17F, IL-21, and TGF-. Dendritic

cells produce multiple cytokines to promote Th17 differentiation, such as IL-6, IL1-, IL-23,

and activin A. Dendritic cells also express integrin v8 to process the latent form of TGF-

to the active form.

Figure 2: TGF- promotes Th17 differentiation through multiple mechanisms

STAT3, induced by IL-6 or IL-21, potentiates the Th17 related transcriptional program by

binding to Rorc, Il17a, and Il21 loci. TGF- receptor signaling phosphorylates SMAD2 and

SMAD3. The SMAD2/3 complex interacts with SMAD4 and translocates into the nucleus.

Meanwhile, SMAD2 and SMAD3 act as a co-activator and co-repressor of STAT3, and

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 oppositely modify STAT3-induced transcription. TGF- signaling triggers SKI degradation to

unleash SMAD4 and SKI complex repressed RORt expression. TGF- suppresses SOCS3
Accepted Article
to prolong STAT3 activation. TGF- suppresses GFI-1 to promote Il17a transcription. TGF-

suppresses Eomes through JNK-c-JUN pathway to enhance Rorc and Il17a transcription.

TGF- drives RhoA-ROCK2 to phosphorylate IRF4 and to upregulate the expression of

RORt, IL-17 and IL-21. TGF- induces TAZ to co-activate RORt and to reduce Foxp3

through proteasomal degradation.

Figure 3: T helper differentiation.

Diagram of T helper differentiation pathways for Th1, Th2, Th9, Th17, Th22, Treg, Tr1, and

Tfh. Cytokines that play an important role in inducing CD4+ T cell differentiation are listed

above the cell types, and cell-specific transcription factors are listed in a box below the cell

types.

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Accepted Article

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Accepted Article

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Accepted Article

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