Prostaglandin E2 (PGE2) signaling pathways in T cells: significance in anti-tumor immune function

Background: Prostaglandin E2 (PGE2) is an abundant prostanoid involved in many physiological processes including gastrointestinal function, reproduction and cardiovascular function. In addition, it is known to play a detrimental role in certain cancer types, in particular colorectal cancer, where its levels are elevated and correlate with disease progression [1].  PGE2 aids cancer progression through several mechanisms, including inhibition of apoptosis, promotion of metastasis, cell invasion, and angiogenesis as well as induction of regulatory T cells (Tregs). PGE2 is also known to have a number of effects on T cell differentiation, proliferation and other functions relevant in cancer and various inflammatory diseases [2]. In particular, the Taskén lab has characterized an inhibitory PGE2-triggered signaling pathway in T cells [3] whereby PGE2 secreted from Tregs, monocytes or tumor cells inhibits T cell receptor (TCR) signaling and thus attenuates anti-tumor immune responses. To learn more about PGEsignaling and function in T cells on a systems level, the lab has used a combined phosphoproteomics and phosphoflow cytometry approach to map PGE2 signaling pathways through each of its four receptors EP1-4. These studies provided global views of the signaling pathways and networks elicited by PGE2 in T cells and identified novel signaling nodes and pathways [4, manuscript in preparation].

 Figure 1: Illustration of the signal network downstream of the 4 receptors for PGE2, EP1-EP4 where each receptor triggers a subset of kinases and a distinct but overlapping phosphorylation pattern.

Current Project: The current project contains two parts, and can therefore be somewhat adapted to the student’s interests. The first part builds on our recently performed phosphoproteomics study which investigated PGE2 signaling pathways and networks in different subtypes of T cells, including CD4, CD8 and Treg cells. In the study, we characterized the signaling pathways initiated through each of the four different PGE2 receptors (EP1-4) present in T cells by using highly specific agonists to trigger each receptor (Figure 1). We then used multiplexed phosphoflow cytometry to perform more detailed analysis of the pathways and networks identified by phosphoproteomics. This has yielded a map of pathways, kinases, and networks that we believe to be active downstream of the receptors (manuscript in preparation).

In the current project, we will first use phosphoflow cytometry in combination with kinase inhibitors to study how the identified pathways behave when perturbed. In particular, we will stimulate each receptor and assess the effect of different kinase inhibitors on the pathways identified. This will help assess the contribution of each kinase to signaling through each receptor. This will be part of an ongoing collaboration with Julio-Saez Rodriguez’ group where we dynamically model the signaling through each receptor and how it responds when different nodes/kinases are inhibited. This will lead to a greater understanding of these pathways and networks connect and interact. We may also use phosphoflow cytometry to look at the interactions between the different receptors, in particular EP2 and EP4, which are thought to signal through some separate and some distinct nodes. If time allows, we will also look at the functional output of EP1-4 signals. In particular, we may look at the effect of stimulation of the different EP receptors, blocking specific kinases with inhibitors and/or knockdown/knock-in of unphosphorylatable mutants of PGE2-regulated phosphoproteins on TCR signaling and/or T cell activation, proliferation, differentiation.

Figure 2. The inhibitory PGE2 pathway in effector T cells. PGE2, through its receptors EP2 or EP4, can trigger an inhibitory pathway that results in the inhibition of T cell receptor (TCR) signaling. We are targeting this pathway at the level of the EBP50-Ezrin interaction, with small molecule inhibitors. This blocks the inhibitory pathway and restores effector T cell function.

The second part of the project deals with blocking the inhibitory PGE2 pathway in T cells (figure 2) by interrupting the EBP50-Ezrin interaction. We have identified molecules that effectively block this interaction in vitro and would now like to test these compounds more comprehensively in vivo, using ELISAs, flow cytometry, and possibly other techniques to assess whether the pathway has been blocked.

These studies should improve the current knowledge of PGE2 signaling networks in different T cell subtypes and may lead to new insights into the functional significance of the different signaling pathways. In the project, the student will gain experience with some of the following techniques: isolation and purification of primary T cells, use of small molecules to probe cellular functions, ELISAs, immunoblotting, phosphoflow cytometry with fluorescent cell barcoding, and possibly knockdown/knock-in functional studies in T cells.

 

  1. Brudvik KW, Henjum K, Aandahl EM, Bjornbeth BA, Tasken K (2012) Regulatory T-cell-mediated inhibition of antitumor immune responses is associated with clinical outcome in patients with liver metastasis from colorectal cancer. Cancer Immunology Immunotherapy 61: 1045-1053.
  2. Lone AM and Tasken K (2013) Proinflammatory and immunoregulatory roles of eicosanoids in T cells. Front Immunol, 4: 130
  3. Vang T, Torgersen KM, Sundvold V, Saxena M, Levy FO, et al. (2001) Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor. Journal of Experimental Medicine 193: 497-507.
  4. Oberprieler NG, Lemeer S, Kalland ME, Torgersen KM, Heck AJR, et al. (2010) High-resolution mapping of prostaglandin E-2-dependent signaling networks identifies a constitutively active PKA signaling node in CD8(+)CD45RO(+) T cells. Blood 116: 2253-2265.

 

Publisert 5. feb. 2019 13:30 - Sist endret 5. feb. 2019 13:30

Omfang (studiepoeng)

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