In Vivo Modeling of Human ?d T Cell Ontogeny Reveals Terminal Deoxynucleotidyl Transferase as a Key Regulator of Type 3 Vd2 T Cell Development

Profound differences in T cell receptor (TCR) repertoire and functional profiles between human and murine ?d T cells pose significant challenges for translational ?d T cell research. Therefore, we generated humanized immune system (HIS) NBSGW (NOD,B6.PrkdcscidIl2r?-/-KitW41/W41) mice reconstituted with human fetal liver CD34+ hematopoietic stem and progenitor cells (HSPCs) enabling human ?d T cells evaluation in vivo. The HIS mice accurately recapitulated the TCR-associated thymic programming of human ?d T cells—alongside aß T cells—and their peripheral effector functions, including the generation of phosphoantigen-reactive V?9Vd2 T cells uniquely found in humans. Moreover, terminal deoxynucleotidyl transferase (TdT) was identified as a key regulator of type 3 Vd2 T cell development. These findings demonstrate that HIS mice are a powerful model to screen human ?d T cell-targeting immunotherapies and to obtain mechanistic insights in human ?d T cell biology. Overall design: ?d and aß T cells were sorted from the thymi and peripheral organs of humanized immune system (HIS) mice transplanted with human HSPCs, followed by 5'-RNA single cell transcriptome, ADT and TCR sequening (10x genomics). In parallel, after knocking-out (KO) DNTT in the input HSPCs of HIS mice by CRISPR, ?d and aß T cells were sorted from the thymi and spleens of control or KO HIS mice, followed by 5'-RNA single cell transcriptome, ADT and TCR sequening (10x genomics).