CZ
Cloé Zamit
  • Ph. D. Candidate, Aix-Marseille Université, CNRS, INSERM, CIML, Centre d'Immunologie de Marseille-Luminy, Marseille, France Marseille
Research fields
  • Immunology
Medullary Thymic Epithelial Cell Antigen-presentation Assays
Authors:  Alexia Borelli, Cloé Zamit and Magali Irla, date: 11/05/2023, view: 583, Q&A: 0

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3+ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4+ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4+ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4+ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4+ T cells in an antigen-specific context.


Key features

• This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022).

• This protocol requires transgenic mice, such as OTIIxRag2-/- mice and the cognate peptide OVA323–339, to assess mTEC antigen presentation to CD4+ T cells.

• This requires specific equipment such as a Miltenyi Biotec AutoMACS® Pro Separator, a BD FACSAriaTM III cell sorter, and a BD® LSR II flow cytometer.


Graphical overview


Single Molecule Tracking Nanoscopy Extended to Two Colors with MTT2col for the Analysis of Cell-Cell Interactions in Leukemia
Authors:  Loriane Maillot, Magali Irla and Arnauld Sergé, date: 04/20/2022, view: 1631, Q&A: 0

Single molecule tracking (SMT) is a powerful technique to study molecular dynamics, and is particularly adapted to monitor the motion and interactions of cell membrane components. Assessing interactions among two molecular populations is classically performed by several approaches, including dual-color videomicroscopy, which allows monitoring of interactions through colocalization events. Other techniques, such as fluorescence recovery after photobleaching (FRAP), Förster resonance energy transfer (FRET), and fluorescence correlation spectroscopy (FCS), are also utilized to measure molecular dynamics.


We developed MTT2col, a set of algorithmic tools extending multi-target tracing (MTT) to dual-color acquisition (https://github.com/arnauldserge1/MTT2col). In this protocol, we used MTT2col to monitor adhesion molecules at the contact between leukemic stem cells and stromal cells, a process involved in cancer resistance to chemotherapy and in relapse. Our dual-color single molecule protocol includes the following steps: (i) labeling molecules of interest with fluorescent probes, (ii) video-acquisition, (iii) analyses using our MTT2col in-house software, to obtain positions and trajectories, followed by (iv) detailed analyses of colocalization, distribution, and dynamic motion modes, according to the issues addressed. MTT2col is a robust and efficient SMT algorithm. Both MTT and MTT2col are open-source software that can be adapted and further developed for specific analyses.


Graphical abstract:



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