Human unconventional T cells currently include well-characterised cell types such as MAIT, iNKT and gd T. These cells are functionally and phenotypically distinct from their conventional CD4 and CD8 counterpart, and increasing evidence reported that they play a variety of roles in both physiological and pathological processes. In addition to the well-known members of the unconventional T cell family, we have recently described an additional subset of T cells characterised by the lack of CD4 and CD8 co-receptors and the expression of a alpha/beta T cell receptor. They are therefore known as double negative alpha beta T cells, or DNT. DNT are a rare T cell population in human blood, and have been implicated in a few human pathological settings, such as autoimmune diseases and cancer. However, their biological relevance is not yet fully understood. One of the main limitations for studying DNT is the current lack of positive markers, either at the RNA or at the protein level. Another challenge is the significant phenotypic and functional overlap present among different subsets of the unconventional T cell family. This implicates that in order to detect true DNT-specific features, all other T cell components need to be analysed simultaneously. Moreover, among the different layers of information missing on human DNT, there is no current data on their ontogeny nor on their tissue localisation. Therefore, here we will perform a comparative analysis of human DNT against any other T cell type, studying their transcriptional, phenotypic and functional profile. To this end, we have designed bulk RNAseq experiments, single cell RNAseq and CITEseq experiments, matched with scTCRseq, to link their transcriptional profile to their TCR clonality. Moreover, to bypass the technical challenge of low cell number present in human tissues, we have set up an in vitro platform to expand DNT by >100 fold. In this project we will leverage such platform to perform both transcriptional profiling and functional studies. Overall we expect to provide a formal definition to human DNT in terms of origin, relation to other cognate T cell populations and functional capacity. Such data will be useful to better understand the contribution of human DNT to several pathological states and to potentially employ DNT as a therapeutic tool in the future.