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Session Overview
Cell Signalling
Time: Friday, 14/Oct/2011: 2:30pm - 3:30pm
Session Chair: Hyun-Dong Chang
Location: Lecture Hall


Quantitative single cell analysis of endogenous transcription factor expression levels reveals NFATc2 and c-fos as limiting factors for IL-2 production

Tobias Scheel1, Hanna Bendfeldt1, Manuela Benary2, Hanspeter Herzel2, Ria Baumgrass1

1Deutsches Rheuma-Forschungszentrum Berlin, Germany; 2Institute for Theoretical Biology, Humboldt University zu Berlin, Germany; scheel@drfz.de

The regulation of Interleukin-2 (IL-2) expression in human Th cells is of great importance for the balance of the immune system: effective immune responses vs. central tolerance. Therefore, precise fine-tuning of IL-2 expression is crucial for adjusting the immune response.

So far it is unknown, whether IL-2 producing and nonproducing Th cells are different in the expression levels and activatability of the main TCR-dependent transcription factors. Using transcription factor analysis on single cell level by flow cytometry and mathematical analysis we studied the transcription factor networks regulating IL-2 production in human memory Th cells. We showed that physiological differences in the expression level of NFATc2 and c-fos, but not of c-jun and NF-κBp65, are limiting for the decision whether IL-2 is expressed or not. Particularly, stochastic variation in the expression of c-fos leads to substantial diversity of IL-2 expression in approximately 40% of the memory Th cells. The remaining cells exhibit a high probability of IL-2 expression, thereby ensuring robustness in IL-2 response within the population. Manipulation of c-fos de novo synthesis by the small molecular inhibitor U0126 confirms the importance of a certain endogenous c-fos expression level for IL-2 production.

These findings reveal how T cells benefit from regulated variation in transcription factor expression to achieve variability of cytokine expression in a controlled manner.

The work was supported by the German Federal Ministry of Education and Research (BMBF; Forsys-Partner) and by the Deutsche Forschungsgemeinschaft (DFG; SFB-TR52).

Incubation of human mobilized peripheral and cord blood and bone marrow cells with anti-hVEGFR2 and anti-hS1P3 alters gene expression of pluripotent cell populations.

Anush Vemir Karapetyan, Ahmed Abdel-Latif

University of Kentucky, United States of America; avkara2@email.uky.edu

Hypothesis: antibodies to certain receptors may cause conformational changes in receptor and activate downstream signal transduction cascade.

Vascular endothelial growth factor receptors (VEGFR) belong to receptor tyrosine kinases.

Binding of vascular endothelial growth factor ( VEGF) to extracellular portion of receptor causes conformational changes in intracellular part of receptor. This event leads to activation of kinase activity of receptor with subsequent signal transduction eventually resulting in alteration of genome response. There are 3 types of VEGF receptors with different affinities to various splices of VEGF molecule. In our experiment we were looking on VEGFR2 expression on lineage negative progenitor cells. As lineage positive we classify cells expressing lymphoid and myeloid surface markers.

Sphingosine 1-phosphate (S1P) receptors belong to G-coupled proteins, also responding and transducing extracellular signals through conformational changes caused by extracellular ligand-receptor interaction. There are 5 known S1P receptors designated as S1P1, 2,3,4,5

By using flow cytometry we examined the expression of VEGFR2, CD105, CD117, CXCR4 in lineage negative CD34+ populations after incubation of white blood cells of mPB, CB and BM with antibodies to human VEGFR2. The population of lineage-CD34+VEGFR2+ cells significantly rises after two days of incubation and the expression of CD105 and CD117 changes on some lineage negative subpopulations.

We also examined the phosphorylation of Akt -kinase after incubation with anti-hS1P3.

The positive change in phosphorylation level of Akt after 10 min of incubation mPB with anti-hS1P3 compared to control demonstrates the activation of S1P3 receptors by its antibody.

By using RT-qPCR we also examined the alterations in gene expression of CB cells after incubation with anti-hVEGFR2 and anti-hS1P3 antibodies for 1, 2 and 3 days.

The expression levels of mRNA of homeodomain proteins Nkx2.5 and NANOG and transcription factors GATA4 and oct 4 were several times altered.

As is known GATA4 and Nkx2.5 are expressed in precardiac cells and are considered to direct the differentiation of progenitor cells to cardiac cells.

Instead NANOG and oct4 are major factors expressed in pluripotent cells and responsible for maintaining pluripotency.

Thus VEGFR2 and S1P3 receptors are involved in differentiation of pluripotent progenitor cells and their activity may be regulated by the antibodies to these receptors.

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