Sensor Function for Butyrophilin 3A1 in Prenyl Pyrophosphate Stimulation of Human Vγ2Vδ2 T Cells

Abstract

Vγ2Vδ2 T cells play important roles in human immunity to pathogens and in cancer immunotherapy by responding to isoprenoid metabolites, such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and isopentenyl pyrophosphate. The Ig superfamily protein butyrophilin (BTN)3A1 was shown to be required for prenyl pyrophosphate stimulation. We proposed that the intracellular B30.2 domain of BTN3A1 binds prenyl pyrophosphates, resulting in a change in the extracellular BTN3A1 dimer that is detected by Vγ2Vδ2 TCRs. Such B30.2 binding was demonstrated recently. However, other investigators reported that the extracellular BTN3A1 IgV domain binds prenyl pyrophosphates, leading to the proposal that the Vγ2Vδ2 TCR recognizes the complex. To distinguish between these mechanisms, we mutagenized residues in the two binding sites and tested the mutant BTN3A1 proteins for their ability to mediate prenyl pyrophosphate stimulation of Vγ2Vδ2 T cells to proliferate and secrete TNF-α. Mutagenesis of residues in the IgV site had no effect on Vγ2Vδ2 T cell proliferation or secretion of TNF-α. In contrast, mutagenesis of residues within the basic pocket and surrounding V regions of the B30.2 domain abrogated prenyl pyrophosphate-induced proliferation. Mutations of residues making hydrogen bonds to the pyrophosphate moiety also abrogated TNF-α secretion, as did mutation of aromatic residues making contact with the alkenyl chain. Some mutations further from the B30.2 binding site also diminished stimulation, suggesting that the B30.2 domain may interact with a second protein. These findings support intracellular sensing of prenyl pyrophosphates by BTN3A1 rather than extracellular presentation.

BTN3A molecules considerably improve Vγ9Vδ2T cells-based immunotherapy in acute myeloid leukemia

“…However, only the B30.2 domain of the BTN3A1 isoform can bind pAg 21 through a positively charged pocket. 22,23 This intracellular binding induced extracellular conformational changes of BTN3A, similar to those observed with 20.1 mAb and resulting Vg9Vd2 T cell activation. 24 Together, these data open new perspectives in Vg9Vd2 T cells-based immunotherapies, with BTN3A appearing as an interesting target to regulate their functions.…”

Vγ9Vδ2 TCR‐activation by phosphorylated antigens requires butyrophilin 3 A1 (BTN3A1) and additional genes on human chromosome 6

“…To identify those human chromosome(s) mandatory for PAg presentation, hybrid cells were cloned and tested for induction of reporter cell stimulation in the presence of 1 nM HMBPP. PCR karyotyping showed that loss of human chromosomes 2,3,7,8,9,10,11,13,17,18,20,21, and X had no effect on PAg-mediated activation of the reporter cells, while cells without Chr6 failed to induce activation of the reporter cells in the presence of HMBPP or zoledronate. For reasons so far unknown, 2 of 6 of the Chr6-bearing hybridoma cell lines stimulate the reporter cells in the presence of HMBPP and zoledronate but not in the presence of 2 mM sec-butylamine.…”

“…Given that BTN3A1 protein loaded with PAg in a cell-free system binds to recombinant Vγ9Vδ2 TCRs [12], we would predict that the missing Chr6-encoded gene(s) relate to cellular functions such as PAg loading of the BTN3A1 molecule or control of its cellsurface distribution or cellular compartmentalization, for which the PAg-binding intracellular B30.2 domain of BTN3A1 might be crucial [8][9][10][11][12]. The colocalization of BTN3 with genes associated with antigen-presenting function might be by coincidence, but is clearly reminiscent of what is seen for peptide-presenting MHC molecules [15].…”

“…BTN3A1 differs from the other members of the BTN3 family (BTN3A2 and BTN3A3) mainly by its intracellular domain [8][9][10], which most recently has been shown to contain a PAg-binding site [10], and in aminobisphophonate-induced membrane distribution. These observations [8,9] and the fact that PAg binding to the extracellular domain of BTN3A1 has not been demonstrated [8][9][10][11] have led to models of PAg-and mAb 20.1-induced Vγ9Vδ2 T-cell activation in which PAg and mAb 20.1 induce changes in surface distribution of BTN3A1. These changes may then result in ligation of Vγ9Vδ2 TCR and subsequent cellular activation, either directly or indirectly by recruitment of unknown Vγ9Vδ2 TCRligands.…”

Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire

“…γδ T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are activated by microbe-and host-derived phosphorylated prenyl metabolites (phosphorylated antigens or "phosphoantigens") derived from the isoprenoid metabolic pathway, the most active of which are microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and host isopentenyl pyrophosphate (IPP) (13). These phosphoantigens recently have been shown to be presented or to be sensed by the butyrophilin BTN3A1 (14)(15)(16). Although phosphoantigen-reactive Vγ9Vδ2 T cells were thought to be restricted to primates, there is recent evidence that Vγ9, Vδ2, and BTN3A1 genes are coconserved across a variety of placental mammals including primates, alpaca, armadillo, sloth, dolphin, dromedary, and orca, but not rodents (17).…”