In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic property of fetal hematopoietic stem and precursor cells (HSPCs) caused by high expression of the RNA-binding protein Lin28b. In conclusion, our data indicate that the human fetal thymus generates, in an HSPC/Lin28b-dependent manner, invariant γδ T cells with programmed effector functions.

Phosphoantigen-reactive Vγ9Vδ2 T cells represent the main innate human γδ T cell subset and dominate the fetal and adult peripheral blood γδ T cell repertoire. It has been hypothesized that adult blood Vγ9Vδ2 T cells find their origin in the fetus like it is established for mouse innate γδ T cells. To address this issue, we analyzed the CDR3 of the TCR of human blood and thymic Vγ9Vδ2 T cells from fetal until adult life. We first identified key differences in the CDR3 repertoire of fetal and adult blood Vγ9Vδ2 T cells, including in CDR3 features important for phosphoantigen reactivity. Next, we showed that most of these key adult CDR3 features were already present in the postnatal thymus and were further enhanced upon selection in vitro by the microbial-derived phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. Finally, we demonstrated that the generation of a fetal-type or adult-type Vγ9Vδ2 CDR3 repertoire is determined by the fetal and postnatal nature of the hematopoietic stem and precursor cell. Thus, our data indicate that fetal blood Vγ9Vδ2 T cells find their origin in the fetal thymus whereas adult blood Vγ9Vδ2 T cells are generated to a large degree independently after birth.