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

Abstract

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 <i>TRG</i> and <i>TRD</i> 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 <i>ZBTB16</i> (encoding PLZF), <i>KLRB1</i>, and <i>KLRC1</i>, but consisted of distinct clusters with unrelated Vγ9Vδ2⁺ TCR clones characterized either by <i>TBX21</i>, <i>FCGR3A</i>, and cytotoxicity-associated gene expression (type 1) or by <i>CCR6</i>, <i>RORC</i>, <i>IL23R</i>, and <i>DPP4</i> 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.