Phosphoantigen-sensitive Vγ9Vδ2 T cells are important responders to infections and malignancy. However, the mechanisms by which phosphoantigens stimulate Vγ9Vδ2 T cells are unclear. Here, we synthesized phosphoantigen prodrugs and used them to demonstrate that intracellular delivery of phosphoantigens is required for their activity. The pivaloyloxymethyl prodrug is the most potent phosphoantigen described to date, with stronger stimulation of Vγ9Vδ2 T cells from human peripheral blood and greater ability to induce lysis of Daudi lymphoma cells relative to the previously most potent compound, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP). We demonstrate high binding affinity between phosphoantigens and the intracellular region of butyrophilin 3A1 (BTN3A1), localized to the PRY/SPRY (B30.2) domain, but also affecting the membrane proximal region. Our findings promote a phosphoantigen prodrug approach for cancer immunotherapy and unravel fundamental aspects of the mechanisms of Vγ9Vδ2 T cell activation.

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL-like motif at the N-terminal of several hypothetical unknown and dense granule proteins. Hemagglutinin (HA)-tagged versions of these uncharacterized proteins show co-localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM.

Studies of aryl phosphonate derivatives of a butyrophilin 3A1 ligand have resulted in identification of a potent stimulant of Vγ9 Vδ2 T cells. This compound, a mixed ester bearing one pivaloyloxymethyl substituent and one 1-naphthyl ester displayed an EC of 0.79 nM as a stimulant of T cell proliferation, and a 9.0 nM EC in an assay designed to measure interferon gamma production. In both assays, this is the most potent butyrophilin ligand prodrug yet reported, and thus it should be a valuable tool for studies of T cell function. Furthermore, mixed aryl/acyloxyalkyl esters may represent a new class of phosphonate prodrugs with high efficacy.

The Leishmania spp. protozoa have an abundant surface metalloprotease called MSP (major surface protease), which in Leishmania chagasi is encoded by three distinct gene classes (MSPS, MSPL, MSPC). Although MSP has been characterized primarily in extracellular promastigotes, it also facilitates survival of intracellular amastigotes. Promastigotes express MSPS, MSPL, and two forms of MSPC RNAs, whereas amastigotes express only MSPL RNA and one MSPC transcript. We confirmed the presence of MSPC protein in both promastigotes and amastigotes by liquid chromatography-tandem mass spectrometry (LC-MS/MS). More than 10 MSP isoforms were visualized in both amastigotes and promastigotes using two-dimensional immunoblots, but amastigote MSPs migrated at a more acidic pI. Promastigote MSPs were N-glycosylated, whereas most amastigote MSPs were not. Immuno-electron microscopy showed that two-thirds of the promastigote MSP is distributed along the cell surface. In contrast, most amastigote MSP localized at the flagellar pocket, the major site of leishmania endocytosis/exocytosis. Biochemical analyses indicated that most amastigote MSP is soluble in the cytosol, vesicles or organelles, whereas most promastigote MSP is membrane-associated and GPI anchored. Activity gels and immunoblots confirmed the presence of a novel proteolytically active amastigote MSP of higher Mr than the promastigote MSPs. Furthermore, promastigote MSP is shed extracellularly whereas MSP is not shed from axenic amastigotes. We conclude that amastigotes and promastigotes both express multiple MSP isoforms, but these MSPs differ biochemically and localize differently in the two parasite stages. We hypothesize that MSP plays different roles in the extracellular versus intracellular forms of Leishmania spp.

Small isoprenoid diphosphates, such as ()-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (STAA or TA) investigated, confirm that the backbone amide of at least one Thr (Thr), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM-C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.