summer research homogenity in th9 cultures 2
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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
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