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.

Significance: Early-life immune responses have been described as suboptimal, with neonates and infants being susceptible to infections. Vγ9Vδ2 T cells are the first T lymphocytes to be generated in the human fetus. Their T cell receptor-mediated responses to in vitro stimulation and their effector functions at birth are weaker compared with those in adults, possibly reflecting the need for tolerance in utero. However, here we show that upon transition to the prominent microbial exposure early after birth, public fetal-derived Vγ9Vδ2 T cells expand and differentiate into potent cytotoxic effector cells. Thus, they provide newborns with a first line of antimicrobial effector T cells in order to combat infections in early life.

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.

Developmental thymic waves of innate-like and adaptive-like γδ T cells have been described, but the current understanding of γδ T cell development is mainly limited to mouse models. Here, we combine single cell (sc) RNA gene expression and sc γδ T cell receptor (TCR) sequencing on fetal and pediatric γδ thymocytes in order to understand the ontogeny of human γδ T cells. Mature fetal γδ thymocytes (both the Vγ9Vδ2 and nonVγ9Vδ2 subsets) are committed to either a type 1, a type 3 or a type 2-like effector fate displaying a wave-like pattern depending on gestation age, and are enriched for public CDR3 features upon maturation. Strikingly, these effector modules express different CDR3 sequences and follow distinct developmental trajectories. In contrast, the pediatric thymus generates only a small effector subset that is highly biased towards Vγ9Vδ2 TCR usage and shows a mixed type 1/type 3 effector profile. Thus, our combined dataset of gene expression and detailed TCR information at the single-cell level identifies distinct functional thymic programming of γδ T cell immunity in human.

Summary: While γδ T cells are present virtually in all vertebrates, there is a remarkable lack of conservation of the TRG and TRD loci underlying the generation of the γδ T cell receptor (TCR), which is associated with the generation of species‐specific γδ T cells. A prominent example is the human phosphoantigen‐reactive Vγ9Vδ2 T cell subset that is absent in mice. Murine γδ thymocyte cells were among the first immune cells identified to follow a wave‐based layered development during embryonic and early life, and since this initial observation, in‐depth insight has been obtained in their thymic ontogeny. By contrast, less is known about the development of human γδ T cells, especially regarding the generation of γδ thymocyte waves. Here, after providing an overview of thymic γδ wave generation in several vertebrate classes, we review the evidence for γδ waves in the human fetal thymus, where single‐cell technologies have allowed the breakdown of human γδ thymocytes into functional waves with important TCR associations. Finally, we discuss the possible mechanisms contributing to the generation of waves of γδ thymocytes and their possible significance in the periphery.