Marshall, DJ; (2013) The Role of Multiple Cell Types In the Development of Regulatory T-cells. Doctoral thesis , UCL (University College London).

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Abstract

FoxP3 expressing regulatory T-cells (Treg) are essential for preventing autoimmunity by the immune system. The dynamics and signalling requirements for Treg development in the thymus are not well understood but are thought to integrate TCR, co-stimulatory and cytokine signalling. Previous studies have been hampered by the difficulty of distinguishing peripheral homeostasis from de novo thymic generation of Treg.To circumvent this problem, we used mice bearing both a FoxP3 reporter allele (FoxP3GFP) and in which Zap70 expression is controlled by a Tet-inducible transgene (TetZap70), induced by administration of antibiotic doxycycine (dox). Zap70 deficient thymocytes are arrested at the CD⁴+CD⁸+ double positive stage of development. Induction of Zap70 expression by dox therefore restores positive selection and allows analysis of de novo Treg development independently of existing peripheral Treg. In timecourses of Zap70 induction of TetZap70 FoxP3GFP mice, we found that Treg develop after day 4 and remained in the thymus until day 10, at which time GFP+ Treg were first detected in peripheral lymphoid organs. To investigate the requirement for TCR signals for Treg development we used a pulse of the tetracycline analog methacycline, which resulted in a tight 48h window of Zap70 induction. Remarkably, confining Zap70 expression to the first two days of thymic development was sufficient for normal development 4 days later. Using the TetZap70 FoxP3GFP mice we also investigated the temporal requirement for TGFβ, IL-2 and CD40 signalling during Treg development. Neither TGFβ nor CD40 signalling were required for de novo thymic Treg development. Using blocking antibodies and the addition of cytokine-antibody complexes revealed an essential role for IL-2 as well as a semi redundant role for IL-15. Blockade of IL-2 had no effect on induction of FoxP3 or the number of Treg induced during development. However, induction of CD25 by FoxP3+ Treg was entirely IL-2 dependant. Using mixed bone marrow chimeras we show evidence supporting a hematopoietic source of thymic IL-2. We therefore propose a model of thymic Treg development in which TCR signals alone are sufficient to induce FoxP3 expression but that continued development of Treg is reinforced by IL-2.

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