Final Activity Report Summary - TREG INVIVO_IMAGING (Cellular interactions in peripheral immune regulation)
Live imaging of lymphocyte motility by intravital microscopy
This aim of this research project is to characterise CD4+Foxp3+ Regulatory T cell (Treg) -related cellular interactions occurring during immune regulatory events. Conventional and multi-photon confocal microscopy will be applied to visualise interactions of APCs with Treg and naïve (effector) T cells both in vitro (suppression assay system) and in vivo by analysing intact organs of different animal models of immune regulation. Here, we present our intravital multi-photon microscopy system that was set-up to visualise concomitantly 3 different cellular subsets during (I) in vitro cultures and (II) real-time in vivo images. We successful managed not only to obtain kinetics and aggregation patterns that are being used to test the prediction of the models in vitro but also we managed develop the conditions to test the models in vivo. The system allows us to perform kinetics of cellular movements and interactions, in various tissues for each single mouse, namely in inguinal, popliteal and cervical LN's and in the skin. In addition, the analysis can be performed at various time points during the course of an immune response/inflammation. We have also developed a semi-automated program to analyse and illustrate cell-velocities, cell-trajectories and cellular interactions occurring between 3 different cell-types within live tissues and during in vitro cultures.
Generation of Treg and development of Animal models
A limiting step in the study of Treg mediated immune regulation in vivo is the low number of Treg available. To perform in vivo real-time imaging of Treg and their target cell population/s, several millions of monoclonal, antigen-specific Treg are required for each mouse. We developed several experimental strategies to promote the generation of Treg in mice that are Transgenic for a T-cell Receptor (TCR) specific for a non-self antigen. These TCR Tg mice are normally devoid of CD4+Foxp3+ Treg. We demonstrate that by altering the cellular composition of the thymus (cells expressing or not expressing the nominal antigen), and thus altering the conditions for negative and positive selection, we induce Treg in high number. We also demonstrate that a single immunisation of the specific peptide emulsified in Complete Freund's Adjuvant (CFA) into the foot-pad of the corresponding TCR transgenic mouse generates high numbers of Treg and prevents spontaneous development of EAE in MBP TCR transgenic mic. These findings are original per se and two manuscripts are being prepared. In addition, these tools allow the purification of peptide-specific Treg and their real-time imaging during active immune and autoimmune reactions.
This aim of this research project is to characterise CD4+Foxp3+ Regulatory T cell (Treg) -related cellular interactions occurring during immune regulatory events. Conventional and multi-photon confocal microscopy will be applied to visualise interactions of APCs with Treg and naïve (effector) T cells both in vitro (suppression assay system) and in vivo by analysing intact organs of different animal models of immune regulation. Here, we present our intravital multi-photon microscopy system that was set-up to visualise concomitantly 3 different cellular subsets during (I) in vitro cultures and (II) real-time in vivo images. We successful managed not only to obtain kinetics and aggregation patterns that are being used to test the prediction of the models in vitro but also we managed develop the conditions to test the models in vivo. The system allows us to perform kinetics of cellular movements and interactions, in various tissues for each single mouse, namely in inguinal, popliteal and cervical LN's and in the skin. In addition, the analysis can be performed at various time points during the course of an immune response/inflammation. We have also developed a semi-automated program to analyse and illustrate cell-velocities, cell-trajectories and cellular interactions occurring between 3 different cell-types within live tissues and during in vitro cultures.
Generation of Treg and development of Animal models
A limiting step in the study of Treg mediated immune regulation in vivo is the low number of Treg available. To perform in vivo real-time imaging of Treg and their target cell population/s, several millions of monoclonal, antigen-specific Treg are required for each mouse. We developed several experimental strategies to promote the generation of Treg in mice that are Transgenic for a T-cell Receptor (TCR) specific for a non-self antigen. These TCR Tg mice are normally devoid of CD4+Foxp3+ Treg. We demonstrate that by altering the cellular composition of the thymus (cells expressing or not expressing the nominal antigen), and thus altering the conditions for negative and positive selection, we induce Treg in high number. We also demonstrate that a single immunisation of the specific peptide emulsified in Complete Freund's Adjuvant (CFA) into the foot-pad of the corresponding TCR transgenic mouse generates high numbers of Treg and prevents spontaneous development of EAE in MBP TCR transgenic mic. These findings are original per se and two manuscripts are being prepared. In addition, these tools allow the purification of peptide-specific Treg and their real-time imaging during active immune and autoimmune reactions.