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Using full spectrum flow cytometry, we designed a 28-color panel for the analysis of markers known to be associated with the γδ T cell immune response. This panel allows the classification of γδ T cell subsets via specific V gene usage (Vγ9, Vδ1, Vδ2, and Vδ3) of their T cell receptor (TCR) and according to their functional differentiation. Phenotypical surface receptors to distinguish different stages of cell maturation included CD45RA, CD27, CD28, CD127, CD57, and CD16; chemokine receptors CXCR6, CCR5, CCR6, and CX3CR1; NK-associated markers NKG2A, NKG2D, CD56, and CD161, checkpoint-inhibitor PD-1, and activating receptors CD38 and CD25. T cell lineage markers for the analysis of αβ T cells (CD4 and CD8) and MAIT cells (Vα7.2) were also included. This optimized multicolor panel allows a comprehensive immune-profiling of all main human γδ T cell subsets and is suitable for longitudinal or exploratory analysis of γδ T cell development and γδ T cell dynamics in clinical cohorts.

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

Accumulating evidence suggests that the mouse embryonic thymus produces distinct waves of innate effector gamma delta 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 gamma delta T cells. Here, we present a protocol for high-throughput sequencing of TRG and TRD pairs that comprise the clonal gamma delta TCR. In combination with single-cell RNA sequencing, multiparameter flow cytometry, and TCR sequencing, we reveal a high heterogeneity of gamma delta T cells sorted from neonatal and adult blood that correlated with TCR usage. Immature gamma delta T cell clusters displayed mixed and diverse TCRs, but effector cell types segregated according to the expression of either highly expanded individual V delta 1(+) TCRs or moderately expanded semi-invariant V gamma 9V delta 2(+) TCRs. The V gamma 9V delta 2(+) 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 V gamma 9V delta 2(+) 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 gamma delta 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 gamma delta T cells with semi-invariant V gamma 9V delta 2(+) TCR develop in the early fetal thymus and persist into adulthood.

Accumulating evidence suggests that the human embryonic thymus produces distinct waves of innate effector γδ T cells. However, it is unclear whether this process comprises a dedicated subset of IL-17-producing γδ T (γδT17) cells, like reported in mice. Here we present a novel protocol for high-throughput paired γδ TCR-sequencing, which in combination with single-cell RNA-sequencing revealed a high heterogeneity of effector γδ T cell clusters. While immature γδ T cell clusters displayed mixed and diverse TCR, effector cell types in neonatal and adult blood segregated according to γδTCR usage. In adult samples, mature Vδ1+ T cells segregated into exhausted PD-1hi and active PD-1low clusters. Among Vγ9Vδ2+ T cell subsets, we identified distinct PLZF-positive effector γδ T cell clusters with innate type-1 and type-3 T cell signatures that were already detectable in a public dataset of early embryonic thymus organogenesis. Together, this suggests that functionally distinct waves of human innate effector γδ T cells including CCR6+ γδT17 cells develop in the early fetal thymus and persist into adulthood.

Starting at birth, the immune system of newborns and children encounters and is influenced by environmental challenges. It is still not completely understood how γδ T cells emerge and adapt during early life. Studying the composition of T cell receptors (TCRs) using next-generation sequencing (NGS) in neonates, infants, and children can provide valuable insights into the adaptation of T cell subsets. To investigate how neonatal γδ T cell repertoires are shaped by microbial exposure after birth, we monitored the γ-chain (TRG) and δ-chain (TRD) repertoires of peripheral blood T cells in newborns, infants, and young children from Europe and sub-Saharan Africa. We identified a set of TRG and TRD sequences that were shared by all children from Europe and Africa. These were primarily public clones, characterized by simple rearrangements of Vγ9 and Vδ2 chains with low junctional diversity and usage of non-TRDJ1 gene segments, reminiscent of early ontogenetic subsets of γδ T cells. Further profiling revealed that these innate, public Vγ9Vδ2(+) T cells underwent an immediate TCR-driven polyclonal proliferation within the first 4 wk of life. In contrast, γδ T cells using Vδ1(+) and Vδ3(+) TRD rearrangements did not significantly expand after birth. However, different environmental cues may lead to the observed increase of Vδ1(+) and Vδ3(+) TRD sequences in the majority of African children. In summary, we show how dynamic γδ TCR repertoires develop directly after birth and present important differences among γδ T cell subsets.

Both, jawless and jawed vertebrates possess three lymphocyte lineages defined by highly diverse antigen receptors: Two T-cell- and one B-cell-like lineage. In both phylogenetic groups, the theoretically possible number of individual antigen receptor specificities can even outnumber that of lymphocytes of a whole organism. Despite fundamental differences in structure and genetics of these antigen receptors, convergent evolution led to functional similarities between the lineages. Jawed vertebrates possess αβ and γδ T-cells defined by eponymous αβ and γδ T-cell antigen receptors (TCRs). "Conventional" αβ T-cells recognize complexes of Major Histocompatibility Complex (MHC) class I and II molecules and peptides. Non-conventional T-cells, which can be αβ or γδ T-cells, recognize a large variety of ligands and differ strongly in phenotype and function between species and within an organism. This review describes similarities and differences of non-conventional T-cells of various species and discusses ligands and functions of their TCRs. A special focus is laid on Vγ9Vδ2 T-cells whose TCRs act as sensors for phosphorylated isoprenoid metabolites, so-called phosphoantigens (PAg), associated with microbial infections or altered host metabolism in cancer or after drug treatment. We discuss the role of butyrophilin (BTN)3A and BTN2A1 in PAg-sensing and how species comparison can help in a better understanding of this human Vγ9Vδ2 T-cell subset.

The T cell receptor (TCR) repertoires of γδ T cells are very different to those of αβ T cells. While the theoretical TCR repertoire diversity of γδ T cells is estimated to exceed the diversity of αβ T cells by far, γδ T cells are still understood as more invariant T cells that only use a limited set of γδ TCRs. Most of our current knowledge of human γδ T cell receptor diversity builds on specific monoclonal antibodies that discriminate between the two major subsets, namely Vδ2+ and Vδ1+ T cells. Of those two subsets, Vδ2+ T cells seem to better fit into a role of innate T cells with semi-invariant TCR usage, as compared to an adaptive-like biology of some Vδ1+ subsets. Yet, this distinction into innate-like Vδ2+ and adaptive-like Vδ1+ γδ T cells does not quite recapitulate the full diversity of γδ T cell subsets, ligands and interaction modes. Here, we review how the recent introduction of high-throughput TCR repertoire sequencing has boosted our knowledge of γδ T cell repertoire diversity beyond Vδ2+ and Vδ1+ T cells. We discuss the current understanding of clonal composition and the dynamics of human γδ TCR repertoires in health and disease.

TCRs of invariant NKT (iNKT) cells bind α-galactosylceramide (αGC) loaded CD1d in a highly conserved fashion and show a characteristic TCR gene usage: An "invariant" α chain with a canonical AV14/AJ18 rearrangement in mice (AV24/AJ18 in humans) is paired with β chains containing characteristic Vβ segments. In the rat, a multimember AV14 gene family increases the variability within this system. This study characterizes CD1d binding of rat AV14 gene segments in TCR transductants as well as CD1d binding and iNKT TCR expression of expanded polyclonal F344 rat iNKT populations. It defines an important role of position 93 at the V-J transition for TCR avidity and species cross-reactivity of the rat iNKT TCR. Furthermore, for the first time we identified variability within the fourth hypervariable loop (HV4) of the α chain as a modulator of CD1d:αGC binding in rat and mouse. Additionally, we confirmed the importance of the CDR2β for CD1d:αGC binding, but also show that the CDR3β may even have opposite effects on binding depending on the pairing α chain. Altogether, we characterized naturally occurring sources of variability for the iNKT TCR and speculate that they rather level than increase the largely germline encoded differences of iNKT TCR ligand avidity.

Summary Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1’s P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner., Graphical Abstract, Highlights • Radiation hybrids identify BTN2A1 as crucial for Vγ9Vδ2 phosphoantigen (P-Ag) sensing • BTN2A1 binds directly to the T cell receptor via germline-encoded regions of Vγ9 • Cell-surface BTN2A1 associates directly with BTN3A1 independent of P-Ag stimulation • The Vγ9-BTN2A1 interaction modality suggests an additional CDR3-dependent TCR ligand, Karunakaran et al. find that butyrophilin 2A1 (BTN2A1) associates with BTN3A1 on the cell surface and binds directly to germline-encoded regions of the Vγ9 chain of the Vγ9Vδ2 TCR. Thus, BTN2A1 collaborates with BTN3A1 to potentiate Vγ9Vδ2 T cell recognition, playing an essential role in phosphoantigen sensing.