Cell-to-cell interaction recording and identification of cellular interactomes that support T cells in the thymus

Our work aims to provide an unbiased resource to study the diversity of the murine thymic stromal compartment in young and aged animals. Understanding how the thymic stroma supporting T-cells changes with age, will help to prevent the decline of adaptative immunity and to improve vaccine responses and tumour surveillance. To tackle this, we have generated a genetic approach to detect cells in physical contact with CD4+ and CD4+CD8+ T cells in vivo in mice: the Yin&Yang mice. The system has two different components: on one hand, inducible sender cells (T cells of interest) that express GFP as a synthetic ligand and, on the other hand, the rest of the cells are receiver cells expressing a synthetic receptor for GFP. Cell-to-cell contact between senders and receivers allows membrane-bound mGFP (expressed by senders) to activate GFP Receptors (expressed by receivers). This activates an orthogonal signaling pathway triggering mCherry expression in the interacting receivers from an inducible reporter gene. Thus, niche cells interacting with sender T cells are fluorescently labeled with mCherry and can be prospectively isolated and characterized by single-cell RNA sequencing. Using a CD4-Cre mouse line we generated CD4+ single positive (SP4) sender cells and a smaller pool of CD4+ CD8+ double positive (DP) sender cells and we performed the transcriptional profiling of isolated mCherry+ niche cells. We coupled this technology with CellPhoneDB analyses to identify relevant molecular pathways regulating the biology of interacting cells.