Intracellular B30 Research Articles

Comprehensive analysis of BTN3A1 in cancers: mining of omics data and validation in patient samples and cellular models.

Butyrophilin 3A1 (BTN3A1), a major histocompatibility complex‐associated gene that encodes a membrane protein with two extracellular immunoglobulin domains and an intracellular B30.2 domain, is critical in T‐cell activation and adaptive immune response. Here, the expression of BTN3A1 in cancers was analyzed in eight databases comprising 86 733 patients of 33 cancers, and the findings were validated in patient samples and cell models. We showed that BTN3A1 was expressed in most cancers, and its expression level was strongly correlated with clinical outcome of 13 cancers. Mutations of BTN3A1 were detected, and the mutations were distributed throughout the entire gene. Gene set enrichment analysis showed that BTN3A1 co‐expression genes and interacting proteins were enriched in immune regulation‐related pathways. BTN3A1 was associated with tumor‐infiltrating immune cells and was co‐expressed with multiple immune checkpoints in patients with breast cancer (BRCA) and non‐small cell lung cancer (NSCLC). We reported that BTN3A1 was downregulated in 46 of 65 (70.8%) NSCLCs, and its expression level was inversely associated with clinical outcome of the patients. BTN3A1 in tumor samples was lower than in counterpart normal tissues in 31 of 38 (81.6%) BRCAs. Bioinformatics analyses showed that BTN3A1 could be a target gene of transcription factor Spi‐1 proto‐oncogene (SPI1), and our ‘wet’ experiments showed that ectopic expression of SPI1 upregulated, whereas silencing of SPI1 downregulated, BTN3A1 expression in cells. These results suggest that BTN3A1 may function as a tumor suppressor and may serve as a potential prognostic biomarker in NSCLCs and BRCAs.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

NLRC5 promotes transcription of BTN3A1-3 genes and Vγ9Vδ2 Tcell-mediated killing.

SummaryBTN3A molecules—BTN3A1 in particular—emerged as important mediators of Vγ9Vδ2 T cell activation by phosphoantigens. These metabolites can originate from infections, e.g. with Mycobacterium tuberculosis, or by alterations in cellular metabolism. Despite the growing interest in the BTN3A genes and their high expression in immune cells and various cancers, little is known about their transcriptional regulation. Here we show that these genes are induced by NLRC5, a regulator of MHC class I gene transcription, through an atypical regulatory motif found in their promoters. Accordingly, a robust correlation between NLRC5 and BTN3A gene expression was found in healthy, in M. tuberculosis-infected donors' blood cells, and in primary tumors. Moreover, forcing NLRC5 expression promoted Vγ9Vδ2 T-cell-mediated killing of tumor cells in a BTN3A-dependent manner. Altogether, these findings indicate that NLRC5 regulates the expression of BTN3A genes and hence open opportunities to modulate antimicrobial and anticancer immunity.

Structure-Activity Relationships of Butyrophilin 3 Ligands.

Phosphoantigens (pAgs) are small phosphorus-containing molecules that stimulate Vγ9Vδ2 T cells with sub-nanomolar cellular potency. Recent work has revealed that these compounds work through binding to the transmembrane immunoglobulin butyrophilin 3A1 (BTN3A1) within its intracellular B30.2 domain. Engagement of BTN3A1 is critical to the formation of an immune synapse between cells that contain pAgs and the Vγ9Vδ2 T cells. This minireview summarizes the structure-activity relationships of pAgs and their implications to the mechanisms of butyrophilin 3 activation leading to Vγ9Vδ2 T cell response.

The Outer Face of the IgV Domain of Butyrophilin 3A1 is Critical for the Sensing of Prenyl Pyrophosphates by Human Vγ2Vδ2 T cells: A New Paradigm for γδ T Cell Recognition

Abstract γδ T cells expressing Vγ2Vδ2 TCR play critical roles in human immunity to microbes and tumors. These cells are uniquely able to use their TCRs for pattern recognition to monitor phosphorylated metabolites in isoprenoid biosynthesis in a process requiring butyrophilin (BTN) 3A1. Metabolites are sensed by binding to the intracellular B30.2 domain and requires the juxtamembrane coiled coil region. The role played by the extracellular domains (ECD) is unclear. To assess the ECD, surface IgV and IgC residues were mutagenized to alanine. Mutant BTN3A1 cDNAs were expressed in 293 T embryonic kidney cells lacking BTN3A1, -A2, and -A3, and the transfected cells pulsed with zoledronic acid and used to stimulate Vγ2Vδ2 T cells. Mutation of 13 residues scattered on the IgC domain had no effect on stimulation. Mutation of 22 residues scattered on the inner, side, and outer faces of the IgV domain also had no effect. However, 4 residues localized to a region on the outer face of the IgV domain either abrogated or diminished stimulation. Others have identified 2 additional residues in this region and mapped recognition to the Vγ2 HV4 loop paralleling BTNL3 and Btnl6 recognition by other γδ TCR. BTN3A2 and BTN3A3 can complement mutant BTN3A1 proteins suggesting that they form heterodimers or can alter their conformation. Modeling of the potential binding interface identifies BTN3 residues that can form ionic or hydrogen bonds to HV4. Given that our previous studies have shown the requirement for all CDRs of Vγ2Vδ2 TCR, a putative second ligand for the conventional binding side is also required. Taken together, we propose that Vγ2Vδ2 T cell stimulation is dependent on the simultaneous binding of BTN3A1 to HV4 and a second ligand to the conventional Vγ2Vδ2 binding site.

Critical Roles for Coiled-Coil Dimers of Butyrophilin 3A1 in the Sensing of Prenyl Pyrophosphates by Human Vγ2Vδ2 T Cells.

Vγ2Vδ2 T cells play important roles in human immunity to pathogens and tumors. Their TCRs respond to the sensing of isoprenoid metabolites, such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and isopentenyl pyrophosphate, by butyrophilin (BTN) 3A1. BTN3A1 is an Ig superfamily protein with extracellular IgV/IgC domains and intracellular B30.2 domains that bind prenyl pyrophosphates. We have proposed that intracellular α helices form a coiled-coil dimer that functions as a spacer for the B30.2 domains. To test this, five pairs of anchor residues were mutated to glycine to destabilize the coiled-coil dimer. Despite maintaining surface expression, BTN3A1 mutagenesis either abrogated or decreased stimulation by (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. BTN3A2 and BTN3A3 proteins and orthologs in alpacas and dolphins are also predicted to have similar coiled-coil dimers. A second short coiled-coil region dimerizes the B30.2 domains. Molecular dynamics simulations predict that mutation of a conserved tryptophan residue in this region will destabilize the dimer, explaining the loss of stimulation by BTN3A1 proteins with this mutation. The juxtamembrane regions of other BTN/BTN-like proteins with B30.2 domains are similarly predicted to assume α helices, with many predicted to form coiled-coil dimers. An exon at the end of this region and the exon encoding the dimerization region for B30.2 domains are highly conserved. We propose that coiled-coil dimers function as rod-like helical molecular spacers to position B30.2 domains, as interaction sites for other proteins, and as dimerization regions to allow sensing by B30.2 domains. In these ways, the coiled-coil domains of BTN3A1 play critical roles for its function.

Probing the Ligand-Binding Pocket of BTN3A1.

Small-molecule phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate stimulate human Vγ9Vδ2 T cells after binding to the intracellular B30.2 domain of the immune receptor butyrophilin 3 isoform A1 (BTN3A1). To understand the ligand-target interaction in greater detail, we performed molecular docking. Based on the docking results, we synthesized the novel ligand (E)-(7-hydroxy-6-methylhept-5-en-1-yl)phosphonate and mutated proposed binding site residues. We evaluated the impact on butyrophilin binding of existing and novel ligands using a newly developed high-throughput fluorescence polarization assay. We also evaluated the ability of the compounds to stimulate proliferation and interferon-γ production of Vγ9Vδ2 T cells. Mutation of H381 fully blocked ligand binding, whereas mutations to charged surface residues impacted diphosphate interactions. Monophosphonate analogs bind similarly to BTN3A1, although they differ in their antigenicity, demonstrating that binding and efficacy are not linearly correlated. These results further define the structure-activity relationships underlying BTN3A1 ligand binding and antigenicity and support further structure-guided drug design.

Towards Deciphering the Hidden Mechanisms That Contribute to the Antigenic Activation Process of Human Vγ9Vδ2 T Cells

Vγ9Vδ2 T cells represent a major unconventional γδ T cell subset located in the peripheral blood of adults in humans and several non-human primates. Lymphocytes that constitute this transitional subset can sense subtle level changes of intracellular phosphorylated intermediates of the isoprenoid biosynthesis pathway (phosphoantigens, pAg), such as isopentenyl pyrophosphate, during cell stress events. This unique antigenic activation process operates in a rigorous framework that requires the expression of butyrophilin 3A1 (BTN3A1/CD277) molecules, which are type I glycoproteins that belong to the B7 family. Several studies have further shown that pAg specifically bind to the intracellular B30.2 domain of BTN3A1 linked to the antigenic activation of Vγ9Vδ2 T cells. Here, we highlight the recent advances in BTN3A1 dynamics induced upon the binding of pAg and the contribution of the different subunits to this activation process. Recent reports support that conformational modifications of BTN3A1 might represent a key step in the detection of infection or tumorigenesis by Vγ9Vδ2 T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that target defined functions of this unique γδ T cell subset.

BTN3A1 Discriminates γδ T Cell Phosphoantigens from Nonantigenic Small Molecules via a Conformational Sensor in Its B30.2 Domain.

Human Vγ9/Vδ2 T-cells detect tumor cells and microbial infections by recognizing small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells; however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag's are discriminated from nonantigenic small molecules. Here, we utilized NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 immunoglobulin variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively charged small molecules, including P-Ag, in a positively charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from nonantigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain and have implications for rational design of antigens for Vγ9/Vδ2-based T-cell immunotherapies.

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

The Juxtamembrane Domain of Butyrophilin BTN3A1 Controls Phosphoantigen-Mediated Activation of Human Vγ9Vδ2 T Cells.

Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1's juxtamembrane domain for correct γδ T cell-related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.

Are the extracellular IgV and IgC domains of butyrophilin 3A1 required for 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 (HMBPP) and isopentenyl pyrophosphate (IPP). The immunoglobulin superfamily protein, butyrophilin 3A1 (BTN3A1), is required for prenyl pyrophosphate stimulation. Our mutational studies and the work of others has shown that HMBPP and IPP compounds are not antigens but instead are sensed molecules that bind to the intracellular B30.2 domain of BTN3A1. How this binding is detected by the Vγ2Vδ2 TCR is unclear. One possibility is that HMBPP binding alters the conformation and/or distribution of the extracellular domains (ECD) of the BTN3A1 dimer allowing direct binding to the Vγ2Vδ2 TCR or to another molecule that binds the Vγ2Vδ2 TCR. Alternatively, the Vγ2Vδ2 TCR could bind to a molecule that is recruited by the intracellular B30.2 domain upon HMBPP binding. To distinguish between these mechanisms, we mutagenized 32 residues on the surfaces of the IgV and IgC domains of BTN3A1 to alanine and tested the mutant proteins for their ability to mediate prenyl pyrophosphate stimulation of Vγ2Vδ2 T cells. Mutagenesis of residues in the IgV and IgC domains had no effect whereas mutagenesis of the B30.2 binding site abrogated Vγ2Vδ2 T cell responses to HMBPP. Therefore, these findings do not support direct binding to the Vγ2Vδ2 TCR (or to another protein) by the ECD of BTN3A1. Instead, Vγ2Vδ2 TCR recognition may be to a protein recruited to the B30.2 domain upon HMBPP binding. Identification of this second protein will help clarify the puzzling process of human Vγ2Vδ2 T cell receptor recognition.

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

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.

Sensor function for butyrophilin 3A1 in prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells (APP5P.100)

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 immunoglobulin superfamily protein, butyrophilin 3A1 (BTN3A1), has been 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 has been recently demonstrated. However, others have 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 binding sites to alanine 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 whereas mutagenesis of residues within the basic pocket and surrounding V regions of the B30.2 domain abrogated prenyl pyrophosphate-induced proliferation and reduced or abolished TNF-α production. Some mutations further from the B30.2 site also diminished stimulation suggesting that the B30.2 domain may interact with a second protein. These findings support intracellular sensing of prenyl pyrophosphates rather than extracellular presentation.

Activation of human γδ T cells by cytosolic interactions of BTN3A1 with soluble phosphoantigens and the cytoskeletal adaptor periplakin.

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 μM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 μM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

Sensing of Pyrophosphate Metabolites by Vγ9Vδ2 T Cells.

The predominant population of γδ T cells in human blood express a T cell receptor (TCR) composed of a Vγ9 (Vγ2 in an alternate nomenclature) and Vδ2 domains. These cells came into the limelight when it was discovered they can respond to certain microbial infections and tumorigenic cells through the detection of small, pyrophosphate containing organic molecules collectively called “phosphoantigens” or “pAgs.” These molecules are intermediates in both eukaryotic and prokaryotic metabolic pathways. Chemical variants of these intermediates have been used in the clinic to treat a range of different cancers, however, directed optimization of these molecules requires a full understanding of their mechanism of action on target cells. We and others have identified a subclass of butyrophilin-related molecules (BTN3A1-3) that are directly involved in pAg sensing in the target cell, leading to engagement and activation of the T cell through the TCR. Our data and that of others support the pAg binding site to be the intracellular B30.2 domain of BTN3A1, which is the only isoform capable of mediating pAg-dependent stimulation of Vγ9Vδ2 T cells. Here, we review the data demonstrating pAg binding to the B30.2 domain and our studies of the structural conformations of the BTN3A extracellular domains. Finally, we synthesize a model linking binding of pAg to the intracellular domain with T cell detection via the extracellular domains in an “inside-out” signaling mechanism of the type characterized first for integrin molecule signaling. We also explore the role of Vγ9Vδ2 TCR variability in the CDR3 γ and δ loops and how this may modulate Vγ9Vδ2 cells as a population in surveillance of human health and disease.

Molecules and Mechanisms Implicated in the Peculiar Antigenic Activation Process of Human Vγ9Vδ2 T Cells.

In human beings, as well as in most non-human primates, the major peripheral γδ T cell subset, which accounts several percent of the whole lymphoid cells pool in adults, carries an heterodimeric TCR composed of Vγ9 and Vδ2 chains. Vγ9Vδ2 T cells are specifically and strongly activated by small organic pyrophosphate molecules termed phosphoantigens (phosphoAg). These low molecular weight compounds are metabolites that are produced by either microbes or endogenously, as intermediates of the mammalian mevalonate pathway, and can accumulate intracellularly during cell stress like transformation or infection. Despite the characterization of numerous natural and synthetic phosphoAg, the mechanism(s) underlying the unique and specific antigenic activation process induced by these compounds remains poorly understood. Activation is both TCR- and cell-to-cell contact-dependent, and results of previous studies have also strongly suggested a key contribution of membrane-associated molecules of primate origin expressed on target cells. The recent identification of B7-related butyrophilin (BTN) molecules CD277/BTN3A, and more precisely their BTN3A1 isoforms, as mandatory molecules in the phosphoAg-induced recognition of target cells by Vγ9Vδ2 T cells opens important opportunities for research and applications in this field. Here, we review the unusual and complex antigenic reactivity of human Vγ9Vδ2 T cells. We highlight the recent advances in our understanding of this process, and propose a model that integrates the type I glycoprotein BTN3A1 and its intracellular B30.2 domain as a physical intermediate implicated in the detection of dysregulated intracellular levels of phosphoAg and the sensing of cell stress by Vγ9Vδ2T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that utilize the unique functional potential of this major γδ T cell subset.

The Intracellular B30.2 Domain of Butyrophilin 3A1 Binds Phosphoantigens to Mediate Activation of Human Vγ9Vδ2 T Cells

In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections, including Mycobacterium tuberculosis, through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical, and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively charged surface pocket. Charge reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that, when introduced into the B30.2 domain of the nonstimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.

Butyrophilin 3A1 Plays an Essential Role in Prenyl Pyrophosphate Stimulation of Human Vγ2Vδ2 T Cells

Most human γδ T cells express Vγ2Vδ2 TCRs and play important roles in microbial and tumor immunity. Vγ2Vδ2 T cells are stimulated by self- and foreign prenyl pyrophosphate intermediates in isoprenoid synthesis. However, little is known about the molecular basis for this stimulation. We find that a mAb specific for butyrophilin 3 (BTN3)/CD277 Ig superfamily proteins mimics prenyl pyrophosphates. The 20.1 mAb stimulated Vγ2Vδ2 T cell clones regardless of their functional phenotype or developmental origin and selectively expanded blood Vγ2Vδ2 T cells. The γδ TCR mediates 20.1 mAb stimulation because IL-2 is released by β(-) Jurkat cells transfected with Vγ2Vδ2 TCRs. 20.1 stimulation was not due to isopentenyl pyrophosphate (IPP) accumulation because 20.1 treatment of APC did not increase IPP levels. In addition, stimulation was not inhibited by statin treatment, which blocks IPP production. Importantly, small interfering RNA knockdown of BTN3A1 abolished stimulation by IPP that could be restored by re-expression of BTN3A1 but not by BTN3A2 or BTN3A3. Rhesus monkey and baboon APC presented HMBPP and 20.1 to human Vγ2Vδ2 T cells despite amino acid differences in BTN3A1 that localize to its outer surface. This suggests that the conserved inner and/or top surfaces of BTN3A1 interact with its counterreceptor. Although no binding site exists on the BTN3A1 extracellular domains, a model of the intracellular B30.2 domain predicts a basic pocket on its binding surface. However, BTN3A1 did not preferentially bind a photoaffinity prenyl pyrophosphate. Thus, BTN3A1 is required for stimulation by prenyl pyrophosphates but does not bind the intermediates with high affinity.

SNP Genotyping and LD Testing in ERMAP: Revealing Scianna Blood Group Diversity in NIH Blood Donors

Abstract 2322 Background:The Scianna blood group system has been implicated in cases of hemolytic disease of the fetus and newborn and the detection of antibodies to rare antigens in this system have impacted on transfusion management in some patients. Recently, it has been discovered that the Scianna blood group antigens are expressed by the erythrocyte membrane-associated protein (ERMAP), a 475 amino acid red cell adhesion protein consisting of 12 exons with the transcription region spanning exons 3–12. Rare variants in exons 4 and 12 have been reported in patients who have made antibodies to Scianna antigens or have a serological null phenotype for the Scianna system. ERMAP is a member of the butyrophilin-like family, featuring an extracellular immunoglobulin variable and intracellular B30.2 domains. Although one ERMAP variant is detected in one commercial molecular assay (Sc1/Sc2), most reported variants in this gene are rare, and therefore remain largely unrecognized during transfusion planning. ERMAP polymorphisms remain unreported on a large scale, contributing to the uncertainty concerning their clinical significance. To fill this void, we characterized seventeen single nucleotide polymorphisms (SNPs) in exons 3, 4, and 12 of ERMAP in 905 repeat blood donors. Methods:The DNA of consenting, repeat NIH blood donors were genotyped for seventeen variants in the ERMAP gene. DNA was isolated from whole blood using the Qiagen's MagAttract EZ1 kit. Following polymerase chain reaction amplification, the samples were genotyped by ligation detection reaction (LDR). LDR utilizes a thermostable ligase to generate single stranded DNA fragments of engineered length with allele-specific fluorescent labels, allowing for rapid, multiplexed genotyping. Ligated products were resolved by capillary electrophoresis (3730 DNA analyzer and GeneMapper software (Life Technologies)). Results:Eleven of the seventeen variants (G35S, R81Q, nt307Δ2, Q296Q, R332X, R392H, L399L, L409L, S442P, L452P, and L452L) were monomorphic in this cohort (N=905). Overall, the 54c>t and 76c>t transitions in exon 3 had minor allele frequencies (MAF) of 0.21 and 0.23, respectively, and appeared in all self-identified ethnic groups (except Native American donors (n=2)) with maxima observed in donors of self-identified Hispanic ethnicity (n=16; MAF=0.41 and 0.44, respectively). These SNPs showed significant linkage disequilibrium (r2=0.86 [95%CI 0.85–0.88]). African-American donors (n=57) had the highest frequency of variant 11c>t (MAF 0.07) and variant 755c>t (MAF 0.018), which was absent or extremely rare in other ethnic groups. The Caucasian donor population was the only group to display variations 788g>a and 1094g>a (MAF 0.003 and 0.0008 respectively). Conclusions:This is the largest sample of blood donors to be comprehensively genotyped for Scianna variants to date. We observed population-specific polymorphism of these rare variants according to the donor’s self-identified ethnicity, which is under further study. Determining the diversity in the Scianna blood group system may help explain otherwise unclear transfusion reactions, particularly if these variants impact on Scianna antigen surface density (especially the predicted leader sequence variants in exon 3) or other ERMAP functions (via variants in the intracellular domain encoded by exon 12). High throughput donor genotyping will allow evaluation of the clinical importance in alloimmunization for variants like the 11c>t, 54c>t, and 76c>t SNPs that lie in the predicted leader sequence and polymorphisms 755c>t, 788g>a, and 1094g>a that lie within the intracellular B30.2 domain of the ERMAP protein. Awareness of the frequencies of these variations can therefore be a clinically useful aid in the investigation of donors implicated in transfusion reactions. Disclosures:No relevant conflicts of interest to declare.