This document summarizes research from the Kaplan Lab on T helper 9 (Th9) cell differentiation and function. The lab found that culturing naive CD4+ T cells under Th9-polarizing conditions (IL-4 and TGFβ) resulted in a homogenous population of IL-9-secreting cells. However, using an IL-9 reporter mouse, they found that the Th9 genetic program was largely intact in IL-9-negative cells, suggesting it is not restricted only to IL-9-secreting cells. The lab aims to better understand Th9 cell differentiation and the roles they play in disease.

1. Homogeneity in Th9
cultures
Kaplan Lab
Daniel Gomes

2. Immunology (background)
Viruses
Bacteria
Fungus
Parasites Fig.1: The innate and adaptive immune response. Cytokines in cancer pathogenesis and cancer therapy Glenn Dranoff Nature Reviews Cancer 4, 11-22 (January 2004)
doi:10.1038/nrc1252

3. Differentiation of Th cells

4. The importance of Th9 cells and IL-9
Th9 Cells in Disease: The Helpful and Unhelpful
Tumor Immunity – May prove effective against tumors such as lung adenocarcinoma.
Helminth Immunity – Clearly provides a powerful mechanism for protection in
helminthic infection such as Trichinella spiralis, Trichuris muris, and Nippostrongylus
brasiliensis.
Inflammatory bowel disease – Recent evidence has found Th9 cells and IL-9 to play
a role in the gut pathology associated with Ulcerative Colitis.
Allergic inflammation– Th9 cells and IL-9 have been found to be critical in both
murine models of asthma, dermatitis, and food allergy.
A greater understanding of Th9 cell differentiation,
stability, and function is important to general health.
Therefore…
Prepared by B. Ulrich Mark H. Kaplan, Matthew M. Hufford and Matthew R. Olson, The development and in vivo function of T helper 9 cells, Nature
reviews Immunology, Vol. 15, pg. 295-305, May 2015.

5. T helper populations are heterogeneous
Naïve Naïve
Naïve
Naïve
Naïve
Naïve
Cultured for 5 days
in Th9 with TGFβ +
IL4 conditions
IL-9
IL-9
IL-9IL-9
IL-9
IL-9
IL-9
IL-9IL-9IL-9
IL-9
IL-9
IL-9
IL-9
IL-9
IL-9
HomogenousHeterogeneous
IL-9
IL-9
IL-9IL-9
IL-9
IL-9
IL-9

6. Is the Th9 genetic Program restricted to Cells
that are secreting IL9?

7. Microarray analysis
of Th9 genetic
program
Jabeen et al., Th9 cell development requires a BATF-regulated transcriptional network, J Clin Invest. 2013 Nov;123(11):4641-53
Genes from
Microarray
data : 344
Genes
from RNA-
seq (GEO)
data : 1778
Il1rl1
Pcdh17
Rab38
Ear10
Lrrc4

8. In-vitro culture of T-helper cells
Harvest Tissue
Spleen
Naïve CD4+ Isolation
Day 0 3 5
T-helper subset specific
Culture Conditions:
Th1 : IL12
Th2 : IL4
Th9 : IL4 and TGFβ
Th17 : IL6 and TGFβ
Harvest
Mouse Naïve CD4+ T cell
Harvested Day 5
cells
TRIzol RNA Isolation
cDNA Synthesis
RT-PCR for Th9 signature transcription
factors, cytokines, and other genes

9. RT-PCR of Day 5 cells TH subsets data
Erg
Transcription
factor
Il17rb
IL-25R
Th1 Th2 Th9 Th17 Th1 Th2 Th9 Th17 Th17Th1 Th2 Th9
Il9
Pcdh17
Proto-cadherin
Th1 Th2 Th9 Th17
Citrine +/wt
Rab38
GTPase activating protein

10. RT-PCR of Day 5 cells TH subsets data
Irf4
Th17Th9Th2Th1
Th17Th9Th2Th1
Lrrc4
Il1rl1
IL-33R Citrine +/wt
Th1 Th2 Th9 Th17
Ear10
Ribonuclease

11. Is the Th9 genetic Program restricted to Cells
that are secreting IL9?

12. Hypothesis:
Th9 genetic program is restricted to IL9
secreting cells.

13. What is an Il9Cit reporter mouse?
WT IL-9 exon 1
CitrineIL-9Cit
ex1
Citrine
Target strategy to insert the citrine reporter gene
at the start codon of the Il9 gene
• IL-9Cit/IL-9Cit is an Il9KO mouse expressing citrine instead of IL-9.
• IL-9Cit/IL-9WT can express both IL-9 and citrine
IL-9
Citrine

14. In-vitro culture of Th9 cells and Il9 detection
Harvest Tissue
Spleen
Naïve CD4+ Isolation
Heterozygous Citrine/WT Mouse Naïve CD4+ T cell
Day 0 3 5
Th9 Culture Conditions: IL-4 (20ng/ml) + TGFβ (2ng/ml)
Sort
RT-PCR for Th9
signature
transcription
factors,
cytokines, and
other genes.
Citrine +
Citrine -
Day 0 3 5
T-helper subset specific
Culture Conditions:
Th2 : IL4
Harvest

15. The difference in expression between the Cit+ and Cit-
populations
Citrine +/wt
Il9 Pcdh17 Il1rl1

16. The difference in expression between the Cit+ and Cit-
populations
Citrine +/wt
Rab38

17. IL-9
Citrine
IL9 Erg Batf Rab38 Il1rl1 Pcdh17
+++ ++ ++ ++ +++ +++
+ ++ ++ ++ + +
Conclusions
The Th9 genetic program is largely intact in Citrine negative cells

18.  Kaplan Laboratory
 Mark H Kaplan, Ph.D.
 Benjamin Joseph Ulrich
 Matthew Olson, Ph.D.
 Rukhsana Jabeen,
Ph.D.
 Nahid Akhtar
 Ana Moreira, Ph.D.
 Purna Krishnamurthy
 Jesus Alex Ocana, Ph.D.
 Byung-hee Koh
 Cal Johnson
Acknowledgements