some information about Th9 cells - subset of T-hepler cells - current and future

1. The symphony of the ninth: the
development and
function of Th9 cells
Pavulraj. S
5246
M.V.Sc., scholar
Veterinary pathology
www.biolegend.com/thelper#thelper

2. 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 ……

3. 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)

4. Vijay K. Kuchroo
Center for neurologic diseases, Brigham
and women's hospital, Harvard medical
school, Boston

5. T-helper cells subsets
 Naïve
CD4+ cell (Th) – encounter specific
antigen, became differentiate into effectors T helper
(Rukhsana Jabeen et al., 2012)
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.,
(Femke Broere et al., 2011)

6. Th 1
APC
T reg
Th 2
Naive
CD4+
T follicular
helper
Th 9
Th 17
(Femke Broere et al., 2011)

7.  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:
IFN-γ and IL-12
Naïve CD4+
Th1 cell
secrete
IFN-γ,
TNF- α,
IL-2
(Femke Broere et al., 2011)

8. 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

9.  Each cytokine lead to induction of transcription
factors that regulate IL-9 production and expression
of other genes associated with Th9 phenotypes
(Rukhsana Jabeen et al., 2012)
 There is no specific marker

10. Th9 cell

11. Role of TGF-β
 Transforming growth factor- β
 PMN, macrophages are important source
 Have role in cell growth, proliferation, differentiation
and apoptosis
(Veldhoen et al., 2008)
 TGF-β signal induce expression of Foxp3 and PU.1
transcription factors
(Rukhsana Jabeen et al., 2012)
 PU.1 directly bind to Il9 promoter

12. 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)

13.  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)

14. 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)

15. 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)

16. Transcriptional network in T9 cells
(Rukhsana Jabeen et al., 2012)

17. PU.1
 ETS family transcription factor (E-twenty six)
(Hua-Chen Chang et al., 2009)
 One of the largest families of transcription factors
 Member of this family was identified as a gene
transduced by the leukemia virus, E26
(Blair et al., 2000)
 Activated by TGF-β
 PU.1 is expressed in Th9 cells in higher amounts than
in Th2 cells
(Rukhsana Jabeen et al., 2012)

18. 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)

19. 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 IL9-producing Th9 cells
 Chromatin-immunoprecipitation (ChIP) analyses
revealed direct IRF4 binding to the Il9 promoter in
Th9 cells
(Marc Veldhoen et al., 2010)

20. GATA 3
 Belongs to the GATA family of transcription factors
 GATA-3 has been shown to promote the secretion of IL9 from Th9 cells
 Not bind to Il9 gene
 Cause STAT 6 dependent repression on Foxp3
(Rukhsana Jabeen et al., 2012)

21. 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)

22. Il9 gene
 Il9 gene is responsible for production of IL-9 from
Th9 cells
 Location of Il9 gene
(Rukhsana Jabeen et al., 2012)
Mouse – chromosome 13(40)
Human – chromosome 5(46)
(Goswami et al., 2011)

23.  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)

24. Difference between Th2 and Th9 cells
Th2 cell
 Primed with IL-4
 Transcription factors are
STAT 6, GATA 3
 Producing IL-4, IL-5, IL10, 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)

25. 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)

26. 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)

27. 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)

28. 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)

30. Receptor sites for IL-9
Function of Th9 cells –IL-9
(Goswami et al., 2011)

31. 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)

32. 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)

33. 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)

34. (Staudt et al., 2010)

35. 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)

36. 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)

37. 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 al., 2012)
(Rukhsana Jabeen et

38. Pathogenicity of Th9 cells
Inflammatory cells entering through the optic
nerve head and infiltrating the retina and
vitreous
Inflammatory cells entering through the
limbus and infiltrating the anterior chamber
Inflammatory cells in and around an engorged
retinal vessel
(Cuiyan Tan et al., 2010)

39. 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)

40. 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)

41. 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 Tbet
 IL-9 promote inflammation by stimulating growth of
hematopoietic cells esp. mast cells
 Th9 cells are capable of promoting autoimmune
inflammation

42. 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

43. 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
Immunopharmacology: 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.

44. 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/NFκB 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.

45. Thank you
for your
attention