Antigen specificity and receptor diversity are hallmarks of B and T cells. Only 1%–5% of all T cells bear a γδ T-cell receptor (TCR), and these cells have features of both innate and adaptive cells. While the antigen presentation process was long ago elucidated for αβ T cells, it has remained enigmatic for γδ T cells. In a recent study by Vavassori and co-workers,1 this major gap in knowledge was addressed and closed.
γδ T cells were first discovered in the 1980s.2 They triggered immense interest then and have done so again in recent years.3,4 There are two types of γδ T cells: those that are found in the epithelia and bear an invariant, tissue-specific γδ TCR and those that circulate in the periphery and have a diverse TCR repertoire. The former are generated exclusively within tight time windows in the fetal thymus, and the latter are generated throughout life. γδ T cells secrete inflammatory cytokines quickly after antigen contact and are pivotal in fighting bacterial infections and killing tumor cells. They are an important source of IL-17 and are thereby also involved in autoimmunity.5
Immunological competence is a vital part of the barrier function of epithelial tissues such as the skin or gut. Mouse skin harbors invariant γδ T cells in the epidermis, whereas these cells are found in the dermis in human skin.6 Apparently, human dermal γδ T cells recognize stress-inducible molecules, such as MICA/B, as well as other ligands. In mice, invariant γδ T cells play an important role in the control of inflammatory skin reactions, in wound healing and in cancer surveillance.7 A key question is whether γδ T cells play a similar stress-surveillance role in human skin and to what extent peripheral skin-homing γδ T cells participate in skin inflammation, for better or worse.
It is well established that αβ T cells recognize peptides in the context of antigen-presenting MHC-I or MHC-II molecules. The structure of the human MHC with its peptide-binding groove was resolved decades ago by the groundbreaking work of Björkman.8 However, pathogens contain typical lipids and other molecules beyond peptides, which differ from their hosts. It is not entirely surprising that such molecules are targets of the immune system. Thus, human peripheral Vγ2δ2 (also called Vγ9δ2) T cells specialize in the recognition of small bacterial or self-phosphoantigens, alkylamines, or synthetic aminobisphosphonates. Many pathogenic bacteria form pyrophosphorylated isoprenoid intermediates such as (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP). Eukaryotic cells produce isopentenyl pyrophosphate (IPP) via the mevalonate pathway (Figure 1a).9 IPPs serve as the basis for the biosynthesis of steroids and cholesterols as well as the formation of lipoproteins by prenylation. Notably, their production is upregulated in situations of stress and infection.10
Both HMBPP and IPP are potent stimulators of human γδ T cells, particularly Vγ2δ2/Vγ9δ2. Preferential stimulation by a common microbial isoprenoid metabolite allows responses to a broad array of pathogens through this pathway.11,12
It has long been known that γδ T cells are not MHC restricted and that they can recognize non-peptide antigens, but it remained unclear whether they bind antigen in a manner similar to αβ T cells. It was unknown whether they required an antigen-presenting molecule, and if so, the type of molecule was also unknown. The study by Vavassori et al.,1 recently published in Nature Immunology, identifies butyrophilin 3A1 as the cell surface-bound antigen-presenting molecule for prenyl derivatives to Vγ9δ2 T cells. Vγ9δ2 T cells are a distinct subset of blood γδ T cells that quickly home into perturbed human skin, e.g., into inflamed psoriatic lesions.13 Vγ9δ2 cells are also found in a variety of skin diseases such as cutaneous leishmaniasis.14 Evidence for a role of butyrophilin 3A1 (CD277) had already been provided from studies using an anti-BTN3A1 antibody to stimulate Vγ2δ2 cells.15,16 The elegant study by Vavassori et al. proceeds to prove this beyond doubt, and moreover, it shows the crystal structure and binding details of prenyls in the BTN3A1 molecule. Butyrophilins, which were originally discovered as a cow's milk protein responsible for fat transport, are now recognized as type 1 membrane proteins and members of the immunoglobulin superfamily. They share structural similarities with B7 costimulatory molecules. To date, 13 genes in the human genome have been identified as BTN genes, and in the mouse, 11 BTN genes are known.
Considering that many human cells can stimulate Vγ9δ2 T cells, Vavassori et al. used a comprehensive genetic approach to search for genes that are non-polymorphic, ubiquitously expressed and required for Vγ9δ2 T-cell stimulation. They used human–mouse hybrid cell lines to identify the relevant human chromosomal region. Deleting parts of chromosomes in such lines, they eventually identified arm p of chromosome 6 as the location of interest. Looking at the gene expression profiles of various stimulatory cell lines, they identified 14 candidate genes and examined the remaining stimulatory capacity after the knockdown of each candidate gene. In the end, only the inhibition of BTN3A1 transcripts resulted in reduced presentation of HMBPP and IPP to human Vγ9δ2 T cells. Transfection experiments confirmed these findings and revealed BTN3A1 as a potent antigen-presenting molecule for HMBPP and IPP. At the same time, they showed that the function of BTN3A1 is not that of a costimulatory molecule, but that of a true antigen-presenting molecule capable of stimulating cell lines, transfected cells and Vγ9δ2 T cells freshly isolated from human blood. The use of antibodies specific for BTN3A1 and recombinant soluble BTN3A1 domains demonstrated that phosphorylated antigens bound to the extracellular portion of BTN3A1, forming the stimulatory complex. Each BTN3A1 molecule bound one IPP or HMBPP molecule only, and the binding affinity was at least an order of magnitude lower than the affinity between MHC and peptide antigens. This might be compensated by the longer duration of interaction, which had a half-life of minutes.
Finally, the group crystallized the BTN3A1–IPP complex. The structure confirmed the immunoglobulin V domain similarity and showed no evidence of homodimerization. A conformational change occurred after IPP binding. The distal domain of BTN3A1 bound the prenyls ‘in a shallow pocket, and formed a platform in which amino acid residues of both antigen and BTN3A1 might interact with the TCR’.1 Vγ9Vδ2 T cells could recognize the IPP or HMBPP molecules in the context of BTN3A1, which facilitates binding of the two proteins. Thus, γδ T-cell binding has the same principle as αβ T-cell binding: fixing the antigen in a groove through non-covalent interactions and corecognition of antigen and antigen-presenting molecules by the TCR domain (Figure 1b). Longer prenyl chains could bind as well, extending the range of molecules that can be presented by BTN3A1. Overall, the stimulatory complex is uniquely suited to serve γδ T cells in their sentinel function.4
Which questions remain? Clearly, the study examined only one type of antigen, prenyl-pyrophosphates, whereas there are many more antigen specificities of γδ T cells. It will be important to determine whether other lipid moieties or antigens from other chemical classes are presented by similar molecules, particularly by other butyrophilin family members. Notably, myelin oligodendrocyte glycoprotein is a BTN family member, which may help to elucidate the process of autoimmune mimicry in multiple sclerosis. IPP is an intermediate of the mevalonate pathway, which generates steroids and cortisol. The fact that IPP, i.e., a self-molecule, is also presented and recognized raises the question of whether a triggering threshold exists when this biochemical pathway is disturbed by stress or infections, leading to recognition of affected cells by γδ T cells in cancer and cellular stress. As butyrophilins were originally thought to be costimulatory molecules, more research is needed to clarify whether costimulation is also needed by γδ T cells to fine-tune or control the response. In any case, the diversity regarding BTN molecules, not all of which have orthologs across species, calls for caution in future translational approaches.17 In conclusion, the demonstration of lipid-antigen presentation opens new perspectives for understanding γδ T cell-based immunosurveillance and its therapeutic potential.
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The research of CE is supported by grants from the Deutsche Forschungsgemeinschaft (DFG ES103/5-1 and 103/6-1).
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Esser, C. A fat story—antigen presentation by butyrophilin 3A1 to γδ T cells. Cell Mol Immunol 11, 5–7 (2014). https://doi.org/10.1038/cmi.2013.46
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