A fetal wave of human type 3 effector γδ cells with restricted TCR diversity persists into adulthood.

Accumulating evidence suggests that the mouse embryonic thymus produces distinct waves of innate effector γδ T cells. However, it is unclear whether this process occurs similarly in humans and whether it comprises a dedicated subset of innate-like type 3 effector γδ T cells. Here, we present a protocol for high-throughput sequencing of and pairs that comprise the clonal γδTCR. In combination with single-cell RNA sequencing, multiparameter flow cytometry, and TCR sequencing, we reveal a high heterogeneity of γδ T cells sorted from neonatal and adult blood that correlated with TCR usage. Immature γδ T cell clusters displayed mixed and diverse TCRs, but effector cell types segregated according to the expression of either highly expanded individual Vδ1 TCRs or moderately expanded semi-invariant Vγ9Vδ2 TCRs. The Vγ9Vδ2 T cells shared expression of genes that mark innate-like T cells, including (encoding PLZF), , and , but consisted of distinct clusters with unrelated Vγ9Vδ2 TCR clones characterized either by , , and cytotoxicity-associated gene expression (type 1) or by , , , and expression (type 3). Effector γδ T cells with type 1 and type 3 innate T cell signatures were detected in a public dataset of early embryonic thymus organogenesis. Together, this study suggests that functionally distinct waves of human innate-like effector γδ T cells with semi-invariant Vγ9Vδ2 TCR develop in the early fetal thymus and persist into adulthood.