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The Sauer Lab

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2.3. Untangling a second messenger conundrum: IP4 establishes a feedback loop of Diacylglycerol production that is essential for positive selection.

TCR engagement results in the assembly of a complicated molecular signaling machinery at sites of TCR stimulation (Fig. 4). This machinery then relays signals into the interior of the cell, which in turn direct the cellular response, such as survival and maturation in positive selection, or cell death in negative selection. We found that if IP4 cannot be made due to lack of ItpkB, important signaling molecules including Itk can not be properly recruited to the cell membrane1. Itk is a key activator of another enzyme, PLCγ1, in T cells. PLCγ1 is important for signaling in many cells, because it generates the second messenger molecules IP3 and diacylglycerol (DAG). Underscoring the importance of IP4 in regulating PLCγ1, we found that defective DAG production is a major cause for the block of positive selection in ItpkB-/- DP cells. This established for the first time that DAG is essential for positive selection. DAG mediates this process at least in part by activating the oncogenic Ras/Erk pathway. Whether IP4 regulation of Ras through DAG plays a role in cancer will be an exciting area for future research.

Biochemical and genetic analyses then unraveled an intricate positive feedback loop downstream of the TCR and originating from IP4 via Itk to PLCγ1 (Fig. 4). The feedback becomes possible, because besides producing DAG, PLCγ1 also generates IP3, which in turn is converted into IP4 by ItpkB. Combining all these results, we present a model where TCR stimulation leads to low-level activation of Itk and its effector PLCγ1, allowing low level production of IP3 and DAG. ItpkB then converts the IP3 into IP4. IP4 in turn recruits more Itk to the membrane, where Itk activates PLCγ1 to full extent. This is required to turn up IP3 and DAG production to the full extent required for positive selection.

itpkB feedback loop

Fig. 4 . Proposed role for ItpkB in a positive feedback loop sustaining TCR-induced DAG production in DP thymocytes undergoing positive selection. TCR stimulation results in the activation of proximal tyrosine kinases (Lck, ZAP-70), phosphorylation of transmembrane adaptor proteins including LAT, assembly of signaling complexes centered at LAT, and low level activation of Itk and PLCγ1. This mediates initial low level hydrolysis of the membrane phospholipid PIP2 into the second messengers IP3 and DAG. IP3 mediates Ca2+ mobilization. TCR-induced activation of ItpkB leads to conversion of IP3 into IP4. IP4 then promotes robust membrane recruitment and activation of Itk, thereby allowing for full activation of PLCγ1 and accumulation of DAG to levels sufficient for membrane recruitment and activation of RasGRP1. This in turn mediates activation of Ras and its downstream target Erk, an essential mediator of thymocyte positive selection2.

Taken together, our findings add an unanticipated level of control to the mechanisms mediating TCR signaling in positive selection and thus represent a significant step forward in our understanding of T cell development. They open several exciting avenues for future research in the fields of T cell development and of signaling in general. Moreover, pharmacological modulation of IP4 production through ItpkB could open exciting avenues towards the development of novel therapies for T cell dependent immune diseases such as rheumatoid arthritis, asthma or immunodeficiency.

Further Reading

  1. Huang, Y.H. et al. Positive Regulation of Itk PH Domain Function by Soluble IP4. Science 316, 886-889. (2007).
  2. Alberola-Ila, J. & Hernandez-Hoyos, G. The Ras/MAPK cascade and the control of positive selection. Immunol Rev 191, 79-96 (2003).