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Effector capabilities of γδ T cells are evident in Plasmodium infection in young and adult individuals, while children are the most vulnerable groups affected by malaria. Here, we aimed to investigate the age-dependent phenotypic composition of Vδ1+, Vδ2+, and Vδ3+ T cells in children living in endemic malaria areas and how this differs between children that will develop symptomatic and asymptomatic Plasmodium falciparum infections. Flow cytometric profiling of naïve and effector peripheral blood γδ T cells was performed in 6 neonates, 10 adults, and 52 children. The study population of young children, living in the same malaria endemic region of Ghana, was monitored for symptomatic vs asymptomatic malaria development for up to 42 weeks after peripheral blood sampling at baseline. For the Vδ2+ T cell population, there was evidence for an established type 1 effector phenotype, characterized by CD94 and CD16 expression, as early as 1 year of life. This was similar among children diagnosed with symptomatic or asymptomatic malaria. In contrast, the proportion of type 2- and type 3-like Vδ2 T cells declined during early childhood. Furthermore, for Vδ1+ and Vδ3+ T cells, similar phenotypes of naïve (CD27+) and type 1 effector (CD16+) cells were observed, while the proportion of CD16+ Vδ1+ T cells was highest in children with asymptomatic malaria. In summary, we give evidence for an established adult-like γδ T cell compartment in early childhood with similar biology of Vδ1+ and Vδ3+ T cells. Moreover, the data supports the idea that type 1 effector Vδ1+ T cells mediate the acquisition of and can potentially serve as biomarker for natural immunity to P. falciparum infections in young individuals from malaria-endemic settings.

About 1–5% of human blood T cells are Vγ9Vδ2 T cells. Their hallmark is the expression of T cell antigen receptors (TCR) whose γ-chains contain a rearrangement of Vγ9 with JP (TRGV9JP or Vγ2Jγ1.2) and are paired with Vδ2 (TRDV2)-containing δ-chains. These TCRs respond to phosphoantigens (PAg) such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is found in many pathogens, and isopentenyl pyrophosphate (IPP), which accumulates in certain tumors or cells treated with aminobisphosphonates such as zoledronate. Until recently, these cells were believed to be restricted to primates, while no such cells are found in rodents. The identification of three genes pivotal for PAg recognition encoding for Vγ9, Vδ2, and butyrophilin (BTN) 3 in various non-primate species identified candidate species possessing PAg-reactive Vγ9Vδ2 T cells. Here, we review the current knowledge of the molecular basis of PAg recognition. This not only includes human Vγ9Vδ2 T cells and the recent discovery of BTN2A1 as Vγ9-binding protein mandatory for the PAg response but also insights gained from the identification of functional PAg-reactive Vγ9Vδ2 T cells and BTN3 in the alpaca and phylogenetic comparisons. Finally, we discuss models of the molecular basis of PAg recognition and implications for the development of transgenic mouse models for PAg-reactive Vγ9Vδ2 T cells.

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.

1–5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a TRGV9/TRGJP rearrangement and a TRDV2 comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that TRGV9, TRDV2, and BTN3 genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca (Vicugna pacos) as species with typical Vγ9Vδ2 TCR rearrangements and currently aim to directly identify Vγ9Vδ2 T cells and BTN3. Other candidates to study this coevolution are the bottlenose dolphin (Tursiops truncatus) and the nine-banded armadillo (Dasypus novemcinctus) with genomic sequences encoding open reading frames for TRGV9, TRDV2, and the extracellular part of BTN3. Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predicted BTN3-like gene homologous to human BTN3A3. The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir for Mycobacterium leprae. In this study, we analyzed the armadillo genome and found evidence for multiple non-functional BTN3 genes including genomic context which closely resembles the organization of the human, alpaca, and dolphin BTN3A3 loci. However, no BTN3 transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functional TRGV9/TRGJP rearrangement via PCR failed. In contrast, complete TRDV2 gene segments preferentially rearranged with a TRDJ4 homolog were cloned and co-expressed with a human Vγ9-chain in murine hybridoma cells. These cells could be stimulated by immobilized anti-mouse CD3 antibody but not with human RAJI-RT1Bl cells and HMBPP. So far, the lack of expression of TRGV9 rearrangements and BTN3 renders the armadillo an unlikely candidate species for PAg-reactive Vγ9Vδ2 T cells. This is in line with the postulated coevolution of the three genes, where occurrence of Vγ9Vδ2 TCRs coincides with a functional BTN3 molecule.

CD1d molecules are MHC class I-like molecules that present glycolipids to iNKT cells. The highly conserved interaction between CD1d:α-Galactosylceramide (αGC) complexes and the iNKT TCR not only defines this population of αβ T cells but can also be used for its direct identification. Therefore, CD1d oligomers are a widely used tool for iNKT cell related investigations. To this end, the lipid chains of the antigen have to be inserted into the hydrophobic pockets of the CD1d binding cleft, often with help of surfactants. In this study, we investigated the influence of different surfactants (Triton X-100, Tween 20, Tyloxapol) on in vitro loading of CD1d molecules derived from four different species (human, mouse, rat and cotton rat) with αGC and derivatives carrying modifications of the acyl-chain (DB01-1, PBS44) and a 6-acetamido-6-deoxy-addition at the galactosyl head group (PBS57). We also compared rat CD1d dimers with tetramers and staining of an iNKT TCR transductant was used as readout for loading efficacy. The results underlined the importance of CD1d loading efficacy for proper analysis of iNKT TCR binding and demonstrated the necessity to adjust loading conditions for each oligomer/glycolipid combination. The efficient usage of surfactants as a tool for CD1d loading was revealed to be species-specific and depending on the origin of the CD1d producing cells. Additional variation of surfactant-dependent loading efficacy between tested glycolipids was influenced by the acyl-chain length and the modification of the galactosyl head group with PBS57 showing the least dependence on surfactants and the lowest degree of species-dependent differences.