The symphony of the ninth: the development and

function of Th9 cells

Pavulraj. S5246

M.V.Sc., scholarVeterinary pathology

www.biolegend.com/thelper#thelper

Outline of the presentation Introduction T-helper cells subsets Pathway towards Th9 differentiation Cytokines and transcription factors involved Function of Th9 cells – IL-9 Role of th9 cells in experimental animal models Obstacles Conclusions Future ……

Introduction

CD4+ T helper cells – obligate regulators of inflammatory diseases

There are different subsets of Th cells like Th1, Th2, Th17, Treg, Tfh

Latest addition to the list – Th9 Secrete IL-9 as signature cytokine

(Rukhsana Jabeen et al., 2012)

Vijay K. Kuchroo

Center for neurologic diseases, Brigham and women's hospital, Harvard medical school, Boston

T-helper cells subsets Naïve CD4+ cell (Th) – encounter specific antigen,

became differentiate into effectors T helper subsets

Characterized by distinct pattern of cytokine secretion and function

Th1 – (IL-2, IFN-γ) immunity to intracellular pathogens Th2–(IL-4, IL-5, IL-6) protection against extracellular

pathogens Th17–(IL-17A, IL-17F, IL-21, IL-22) resistance to bacterial

and fungal infection Another effectors subset – Th9 – secrete IL-9, involved in

autoimmunity, asthma, etc.,

(Rukhsana Jabeen et al., 2012)

(Femke Broere et al., 2011)

Th 2

Th 9

Th 17

T follicular helper

T reg

Th 1

Naive CD4+

APC

(Femke Broere et al., 2011)

Ag- activated T cell differentiate in to different phenotype depending on the cytokines in the local environment

Characterized by their cytokine profile and by transcription factors

Eg:

(Femke Broere et al., 2011)

Naïve CD4+ IFN-γ and IL-12

Th1 cell

secrete IFN-γ, TNF- α,IL-2

Pathway towards Th9 differentiation

Naïve Th cells secreting IL-9 are primed in response to cytokines TGF-β and IL-4 are termed as Th9 cells

Both signals are required (Rukhsana Jabeen et al., 2012)

TGF-β and IL-4

IL-9

Each cytokine lead to induction of transcription factors that regulate IL-9 production and expression of other genes associated with Th9 phenotypes

There is no specific marker (Rukhsana Jabeen et al., 2012)

Th9 cell

Role of TGF-β Transforming growth factor- β PMN, macrophages are important source Have role in cell growth, proliferation, differentiation

and apoptosis

TGF-β signal induce expression of Foxp3 and PU.1 transcription factors

PU.1 directly bind to Il9 promoter

(Veldhoen et al., 2008)

(Rukhsana Jabeen et al., 2012)

Role of IL-4 Secreted from Th2 cells and mast cells

It activate the transcription factor STAT6 (signal transducer and activator of transcription 6)

STAT-6 indirectly act regulation of additional transcription factors like IRF4, GATA 3

IRF4 bind to Il9 gene (Rukhsana Jabeen et al., 2012)

IL-4 and STAT 6 repress the Foxp3 and T-bet (both induce T-reg phenotype and suppress IL-9 production)

GATA 3 – master regulator of Th2 cell. Not bind to Il9 gene. Have STAT 6 dependent repression of Foxp3

(Rukhsana Jabeen et al., 2012)

Other cytokines

• IL-25 and IL-1 activate transcription factor NF-κB which bind to Il9 promoter and increase IL-9 production

• IFN–γ and IL-27 inhibit IL-9 production in dose dependent manner

(Rukhsana Jabeen et al., 2012)

Transcription factors for Th9 cells PU.1 – ETS family transcription factor STAT 6 IRF 4 GATA 3 NF –κB

Other cytokines IL-25 IL-1

(Rukhsana Jabeen et al., 2012)

Transcriptional network in T9 cells

(Rukhsana Jabeen et al., 2012)

PU.1 ETS family transcription factor (E-twenty six)

One of the largest families of transcription factors

Member of this family was identified as a gene transduced by the leukemia virus, E26

Activated by TGF-β PU.1 is expressed in Th9 cells in higher amounts than

in Th2 cells(Rukhsana Jabeen et al., 2012)

(Hua-Chen Chang et al., 2009)

(Blair et al., 2000)

STAT 6 Signal transducer and activator of transcription 6

In response IL-4, STAT6 phosphorylated by the receptor associated kinases, and then form homo- or hetero-dimers that translocate to the cell nucleus where they act as transcription activators

(ShreevratGoenka et al., 2000)

IRF 4 Interferon Regulatory Factor 4 Proteins which regulate transcription of interferons IRF4 is also crucial for the development and function

of an interleukin-9 (IL-9)-producing CD4(+) T cell subset Th9

IRF4-deficient CD4(+) T cells failed to develop into IL-9-producing Th9 cells

Chromatin-immunoprecipitation (ChIP) analyses revealed direct IRF4 binding to the Il9 promoter in Th9 cells

(Marc Veldhoen et al., 2010)

GATA 3

Belongs to the GATA family of transcription factors

GATA-3 has been shown to promote the secretion of IL-9 from Th9 cells

Not bind to Il9 gene Cause STAT 6 dependent repression on Foxp3

(Rukhsana Jabeen et al., 2012)

NF-κB Nuclear factor -kappaB

Protein complex that controls transcription of DNA

Resting cell- NF-κB and IκB bounded

On activation IκB destroyed and move to nucleus

Activated by IL-1 (Jacobs, et al., 2012)

Il9 gene

Il9 gene is responsible for production of IL-9 from Th9 cells

Location of Il9 gene

Mouse – chromosome 13(40)Human – chromosome 5(46)

(Rukhsana Jabeen et al., 2012)

(Goswami et al., 2011)

Naïve CD4+ T cell from PU.1 deficient mice fail to produce IL-9

Naïve CD4+ T cell from IRF4 deficient mice fail to produce IL-9

Initially it was thought IL-9 produced by Th2 cells, but now it clearly established that it was produced by Th9 cells

(Rukhsana Jabeen et al., 2012)

Difference between Th2 and Th9 cells

Th2 cell Primed with IL-4 Transcription factors are

STAT 6, GATA 3

Producing IL-4, IL-5, IL-10, IL-13

Markers are CD119 , CD183, CD193

Th9 cell Primed with IL-4 and TGF-β Transcription factors are

STAT 6, GATA 3, PU.1, IRF4, NF –κB

Producing IL-9

No specific

(Femke Broere et al., 2011)

In human

CD4+ T cell acquire Th9 phenotype when differentiate in the presence of TGF-β and IL-4

Other cytokines like IFN-α, IFN-β and IL-21 are potent enhancer of IL-9 production

Blocking IL-21 - ↓IL-9 production Human T cell require PU.1 and IRF4 for expression of

IL-9(Rukhsana Jabeen et al., 2012)

Function of Th9 cells

Th9 cells are pro inflammatory - tending to cause inflammation

Role in broad spectrum of autoimmune disease and allergic inflammation including asthma

Their function is depend on tissue microenvironment and other T-helper cell cytokines

(Rukhsana Jabeen et al., 2012)

Brief about IL-9 14 kDa peptide

Initially called as P40, Mast cell growth factor

Pleiotropic functions in immune system

Associated with allergy and immunity to extracellular parasites

(Goswami et al., 2011)

IL-9 receptor signaling

IL-9 activates a heterodimeric receptor that consists of the IL-9 receptor α-chain (IL-9Rα) and the γ-chain and promotes the cross-phosphorylation of Janus kinase 1 (JAK1) and JAK3

This leads to the activation of signal transducer and activator of transcription 1 (STAT1), STAT3 and STAT5 and the upregulation of IL-9-inducible gene transcription

(Goswami R, et al., 2011)

Receptor sites for IL-9

Function of Th9 cells –IL-9

(Goswami et al., 2011)

Th9 role in autoimmune disease models

T Cell Transfer Model of Colitis

Th9 received mice produce sever colitis, lost weight

(Rukhsana Jabeen et al., 2012)

Experimental autoimmune encephalitis

MOG specific Th9 received mouse developed sever EAE and lesions in CNS

Cells in the CNS of Th9 recipient retained IL-9 producing capacity

IL-9 has role of growth factor of mast cells Treatment with anti-IL-9 antibody ameliorate EAE

(Rukhsana Jabeen et al., 2012)

Role in allergy IL-9 is highly expressed in the lungs of asthmatic

patients IL-9 production was significantly higher in T cell from

atopic infants than non-atopic In mice – transgenic expression of IL-9 in lungs

indicate asthma like response(Rukhsana Jabeen et al., 2012)

(Staudt et al., 2010)

Allergy cont.. Th9 cell ↑ asthma signs characterized by increase in

goblet cell metaplasia, ↑eosinophil infiltration Anti IL-9 antibody result in amelioration of

inflammation

(Staudt et al., 2010)

Role in parasite expulsion Experimentally mice was infected with nematode

Trichuris muris When IL-9 given to mice which cause intestinal

muscle hyper-contractility, facilitating worm expulsion

Mast cells is target for IL-9 growth factor activity in these nematode infection models, and mast cell mediators cause eosinophilia, mucus production, increased intestinal permeability and contractility, which leads to parasite expulsion

(Rukhsana Jabeen et al., 2012)

But…..However, the role of Th9 cell may not be

universal in all models Because in adoptive transfer model, Th9 cells specific

for hen egg lysozyme, transferred into recipient mice expressing HEL in the eye lens

Even though ocular inflammation was developed, cells recovered from inflamed site produced IFN-γ

Anti-IL-9 did not protect the mice May be due to flexibility in cytokine production

(Rukhsana Jabeen et al., 2012)

Pathogenicity of Th9 cells

Inflammatory cells entering through the optic nerve head and infiltrating the retina and vitreous

Inflammatory cells entering through thelimbus and infiltrating the anterior chamber

Inflammatory cells in and around an engorged retinal vessel (Cuiyan Tan et al., 2010)

IL-9 from bench to bed side

As we know now IL -9 expression is more in asthmatic lung, ragweed pollen and other allergic patients

So, blocking antibodies to IL-9 being developed as therapy for atopic diseases

(Goswami et al., 2011)

Obstacles

However, the role of Th9 cell may not be universal in all models

Mechanism of Th9 cell contributing autoimmune and allergic inflammation are distinct has not been examined, is focus of ongoing investigations

IL-9 capable of promote inflammation, but Th9 is the source ?????

(Rukhsana Jabeen et al., 2012)

Conclusions Th9 develop in response to combined signal from TGF-

β and IL-4 Transcription network regulate Th9 development

include TGF- β induced PU.1 IL-4 induced STAT 6 →IRF4, GATA3 & repress Foxp3 and T-bet

IL-9 promote inflammation by stimulating growth of hematopoietic cells esp. mast cells

Th9 cells are capable of promoting autoimmune inflammation

Future …….. But still Th9 & IL-9 has been understudied. Recent

advances in defining Th9 and IL-9 will likely promote greater attention

Next steps in the area will be define the orchestration of Th9 cells and direction by Th9 cells in symphony of inflammation

References • www.biolegend.com/thelper#thelper• Rukhsana, J., and Kaplan, M.H. (2012). The symphony of the ninth: the

development and function of Th9 cells. Current Opinion in Immunology, 24: 303–307.

• Broere, F., Apasov, S.G., Sitkovsky, M.V. and van Eden, W. (2011). T cell subsets and T cell-mediated immunity. Principles of Immuno-pharmacology: 3rd revised and extended editon, Springer Basel AG.

• Veldhoen, M., Uyttenhove, C., van Snick, J., Helmby, H., Westendorf, A., Buer, J., Martin, B., Wilhelm, C., and Stockinger, B. (2008). Transforming growth factor-beta ‘reprograms’ the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nature Immunology, 9:1341-1346.

• Chang, H., Han, L., Jabeen, R., Carotta, S., Nutt, S.L. and Kaplan, M.K. (2009). PU.1 regulates TCR expression by modulating GATA-3 activity. Journal of Immunology, 183: 4887–4894.

• Blair, D.G. and Athanasiou, M. (2000). Ets and retroviruses - transduction and activation of members of the Ets oncogene family in viral oncogenesis. Oncogene, 19: 6472–6481.

References cont..• Goenka, S. and Kaplan, M.H. (2011). Transcriptional regulation by STAT6.

Immunologic Research, 50: 87-96.• Veldhoen, M. (2010). Interferon Regulatory Factor 4: Combinational Control of

Lymphocyte Differentiation. Journal of Immunology.• Jacobs, M.D. and Harrison, S.C. (1998). Structure of an IκBalpha/NF-κB

complex. Cell, 95: 749-758.• Goswami R, Kaplan MH: A brief history of IL-9. J Immunol 2011, 186:3283-

3288.• Cuiyan Tan, Mehak K. Aziz, Jenna D. Lovaas, Barbara P. Vistica, Antigen-Specific

Th9 Cells Exhibit Uniqueness in Their Kinetics of Cytokine Production and Short Retention at the Inflammatory Site, 2010, doi:10.4049/jimmunol

• Staudt V, Bothur E, Klein M, Lingnau K, Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells Immunity. 2010 ;33(2):192-202.

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