Homogeneity in Th9 cultures

Kaplan Lab Daniel Gomes

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

Differentiation of Th cells

The importance of Th9 cells and IL-9Th9 Cells in Disease: The Helpful and UnhelpfulTumor 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.

T helper populations are heterogeneous

Naïve NaïveNaïve

NaïveNaïve

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-9IL-9

IL-9

HomogenousHeterogeneous

IL-9

IL-9

IL-9IL-9

IL-9

IL-9

IL-9

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

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

Il1rl1Pcdh17Rab38 Ear10Lrrc4

In-vitro culture of T-helper cells

Harvest Tissue

Spleen

Naïve CD4+ Isolation

Day 0 3 5

T-helper subset specific Culture Conditions:

Th1 : IL12Th2 : IL4Th9 : IL4 and TGFβTh17 : IL6 and TGFβ

Harvest

Mouse Naïve CD4+ T cell

Harvested Day 5 cells

TRIzol RNA IsolationcDNA Synthesis

RT-PCR for Th9 signature transcription factors, cytokines, and other genes

RT-PCR of Day 5 cells TH subsets dataErg

Transcription factor

Il17rbIL-25R

Th1 Th2 Th9 Th17 Th1 Th2 Th9 Th17 Th17Th1 Th2 Th9

Il9

Pcdh17Proto-cadherin

Th1 Th2 Th9 Th17

Citrine +/wt

Rab38GTPase activating protein

RT-PCR of Day 5 cells TH subsets dataIrf4

Th17Th9Th2Th1

Th17Th9Th2Th1

Lrrc4

Il1rl1IL-33R Citrine +/wt

Th1 Th2 Th9 Th17

Ear10Ribonuclease

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

Hypothesis:Th9 genetic program is restricted to IL9

secreting cells.

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-9Citr

ine

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)

SortRT-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

The difference in expression between the Cit+ and Cit- populations

Citrine +/wtIl9 Pcdh17 Il1rl1

The difference in expression between the Cit+ and Cit- populations

Citrine +/wtRab38

IL-9

Citr

ine

IL9 Erg Batf Rab38 Il1rl1 Pcdh17

+++ ++ ++ ++ +++ +++

+ ++ ++ ++ + +

Conclusions

The Th9 genetic program is largely intact in Citrine negative cells

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