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

Innate-like human type 3 effector γδ T cells with canonical Vγ9Vδ2+ TCR develop in the earliest fetal thymus and persist into adulthood. Early emergence γδ T cells are an innate-like subset of T cells that can recognize and respond to microbes. Tan et al. studied the activation and differentiation of γδ T cells to better understand the development and persistence of these cells. They used single-cell RNA sequencing and paired γδTCR analysis from neonatal cord blood or adult peripheral blood and observed a high level of heterogeneity that correlated with TCR usage in immature and differentiated γδ T cell clusters. They detected type 1– and type 3–like Vγ9Vδ2+ T cell subsets with distinct sets of TCR clonotypes, and similar type 3 Vγ9Vδ2+ T cells were found in neonatal cord blood and the early fetal thymus, suggesting that these cells emerge early in fetal development and can persist 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 TRG and TRD 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 ZBTB16 (encoding PLZF), KLRB1, and KLRC1, but consisted of distinct clusters with unrelated Vγ9Vδ2+ TCR clones characterized either by TBX21, FCGR3A, and cytotoxicity-associated gene expression (type 1) or by CCR6, RORC, IL23R, and DPP4 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.