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A major mechanism of acquired resistance to immune checkpoint inhibition involves downregulation of antigen presentation, including the MHC I complex itself. Downregulation of antigen presentation on MHC I can render tumor cells invisible to any α/β T cell directed therapy. γδ T cells (γδT) are a small subset of the overall T cell compartment, but are characterized by increased cytolytic capacity relative to α/β T cells. Rather than MHC I, γδT recognize target cells via a complex of heterodimerized butyrophilin (BTN) proteins. Thus, display of BTN heterodimers on the surface of tumor cells may enhance immunity to tumors that have downregulated MHC I, or which express low abundance or low affinity antigens. We have previously reported the generation of a heterodimeric BTN protein targeting the CD19 antigen, referred to as BTN2A1/3A1-Fc-CD19scFv. While the BTN2A1/3A1 complex is a potent activator of γδT expressing the Vγ9δ2 T cell receptor (TCR), which is the major γδT population in human peripheral blood, Vγ9δ2 T cells are not the predominant γδT in many tissues. The murine equivalent of BTN2A1/3A1-Fc-CD19scFv, BTNL1/6-Fc-CD19scFv, stimulated specific proliferation and increased the cytolytic capacity of peripheral blood γδT in mice, but not other tissue-restricted GDT populations. These data suggested that distinct BTN heterodimers may preferentially activate tissue-restricted subsets of GDT. To characterize potential differences between peripheral blood and tissue-restricted γδT , we performed a multi-layered analysis, including single-cell sequencing of γδ TCR from paired peripheral blood and tumor tissues from human cancer patients such as melanoma, prostate and colon cancer. These data identified tissue-specific preferences for individual γδ TCRs, with corresponding tissue-specific preferences for individual BTN proteins. Based on this information, we generated a panel of distinct heterodimeric BTN proteins, and show that specific γδT populations are preferentially activated by specific BTN heterodimers in a lock-and-key fashion. These data are a necessary pre-requisite for designing γδT specific therapeutics that may target both immune neglected and acquired resistant tumors that have limited visibility to α/β T cell directed approaches. Citation Format: Suresh de Silva, George Fromm, Anne Lai, Louis Gonzalez, Arpita Patel, Kyung Jin Yoo, Kellsey Johannes, Kinsley Evans, Keith Wilson, Taylor H. Schreiber. Antigen-specific targeting of tissue-resident gamma delta T cells with recombinant butyrophilin heterodimeric fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1736.

Ultrafiltration (UF) is an effective method for separating and enriching milk proteins. This study aims to analyze the protein profile of milk fat globule membrane (MFGM) materials enriched by UF membranes from butter serum with different molecular mass cutoffs through a label-free proteomic approach. In total, 616 MFGM proteins were identified in all groups, of which 395, 399, 399 and 484 proteins were identified with a 30 kDa, 50 kDa and 100 kDa UF membrane and without UF, respectively. It was more efficient to enrich fatty acid binding protein and mucin 15 using a 30 kDa membrane, whereas mucin 1 was more abundant when using a 50 kDa membrane. Compared with the 30 kDa and 50 kDa membranes, the 100 kDa membrane led to the optimal efficiency with low permeation of the most major MFGM proteins and high permeation of non-MFGM proteins. Butyrophilin, adipophilin, xanthine oxidase/dehydrogenase, and periodic acid Shiff6/7 were concentrated approximately twofold in the 100 kDa retentate. Meanwhile, the relative abundances of β-lactoglobulin and α-lactalbumin were reduced by approximately 17.3% and 16.2%, respectively. This work will facilitate the development of MFGM protein isolation using membrane separation processes and provide a theoretical reference for the production of MFGM proteins.

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 EC50 of 0.79 nM as a stimulant of T cell proliferation, and a 9.0 nM EC50 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.

Small isoprenoid diphosphates, such as (E)-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 (ST296AA or T304A) investigated, confirm that the backbone amide of at least one Thr (Thr304), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr296/297) 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 POM2-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.

Exosomes are 40–100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30–100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them. The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of major protein number identified in exosomal preparations after gel filtration may be important to the studies of biological functions of pure exosomes., Graphical abstract Image 1, Highlights • Exosomes are 40–100 nm nanovesicles containing RNA and different proteins. • Well-purified exosomes were obtained using several standard centrifugations and gel filtration. • Well-purified exosomes contained only from five to eight different major proteins. • A possible number of exosome proteins found previously using crude preparations may be much overestimated. • This result may be important to the studies of biological functions of pure exosomes.

Aim Aim of the study was to study the relative gene expression of genes associated with fatty acid synthesis at 60 days postpartum (pp) in bovine mammary epithelial cells (MECs) of Surti and Jafarabadi buffaloes. Materials and methods A total of 10 healthy Surti and Jafarabadi buffaloes of each breed were selected at random from Livestock Research Station, Navsari and Cattle Breeding Farm, Junagadh, Gujarat, respectively, for this study. Milk sample was collected from each selected buffalo at day 60 pp from these two breeds to study relative gene expression of major milk fat genes using non-invasive approach of obtaining primary bovine MECs (pBMEC) from milk samples. Results In this study overall, the relative expression of the six major milk lipogenic genes butyrophilin subfamily 1 member A1 (BTN1A1), stearoyl-CoA desaturase (SCD), lipoprotein lipase (LPL), glycerol-3-phosphate acyltransferase mitochondrial (GPAM), acetyl-coenzyme A carboxylase alpha (ACACA), and lipin (LPIN) did not show changes in expression patterns at 60th day of lactation in both Surti and Jafarabadi buffaloes. Conclusion The pBMEC can be successfully recovered from 1500 ml of milk of Surti and Jafarabadi buffaloes using antibody-mediated magnetic bead separation and can be further used for recovering RNA for down step quantification of major milk lipogenic gene expression. The relative expression of the six major milk lipogenic genes BTN1A1, SCD, LPL, GPAM, ACACA, and LPIN did not show changes in expression patterns in both Surti and Jafarabadi buffaloes, suggesting expression levels of lipogenic genes are maintained almost uniform till peak lactation without any significant difference.

Butyrophilin 3A1 (BTN3A1) binds small phosphorous-containing molecules, which initiates transmembrane signaling and activates butyrophilin-responsive cells. We synthesized several phosphinophosphonates and their corresponding tris-pivaloyloxymethyl prodrugs and examined their effects on BTN3A1. An analog of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) bound to BTN3A1 with intermediate affinity, which was enthalpy-driven. Docking studies revealed binding to the basic surface pocket and interactions between the allylic hydroxyl group and the BTN3A1 backbone. The phosphinophosphonate stimulated proliferation of Vγ9Vδ2 T cells with moderate activity (EC50 = 26 µM). Cellular potency was enhanced >600-fold in the tris-POM prodrug (EC50 = 0.041 µM). The novel prodrug also induced T cell mediated leukemia cell lysis. Analysis of dose response data reveals HMBPP-induced Hill coefficients of 0.69 for target cell lysis and 0.68 in interferon secretion. Together, tris-POM prodrugs enhance the cellular activity of phosphinophosphonates, reveal structure-activity relationships of butyrophilin ligands, and support a negatively cooperative model of cellular butyrophilin activation.

Summary Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, “LES” with an affinity (∼15–25 μM) comparable to many αβ TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined., Graphical Abstract, Highlights • BTNL3 binds directly and specifically to Vγ4+ TCRs via its IgV domain • The superantigen-like binding mode focuses on germline-encoded TCR regions • In contrast, γδ TCR binding to a clonally restricted antigen is CDR3-mediated • Mutagenesis indicates parallels with BTN3A1-mediated activation of Vγ9Vδ2 T cells, Butyrophilin (BTN) and butyrophilin-like (BTNL) molecules powerfully influence selection and activation of specific γδ lymphocyte subsets, but whether they directly bind the γδ TCR has remained contentious. Willcox et al. show that BTNL3 directly binds to human Vγ4+ TCRs via a superantigen-like binding mode that is focused on germline-encoded TCR regions.

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, “LES” with an affinity (∼15–25 μM) comparable to many αβ TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined. Butyrophilin (BTN) and butyrophilin-like (BTNL) molecules powerfully influence selection and activation of specific γδ lymphocyte subsets, but whether they directly bind the γδ TCR has remained contentious. Willcox et al. show that BTNL3 directly binds to human Vγ4+ TCRs via a superantigen-like binding mode that is focused on germline-encoded TCR regions.

Invariant Vγ9Vδ2 T cells respond to "phosphoantigen" metabolites through binding to the B30.2 domain of butyrophilin BTN3A. Yang et al. (2019) use molecular dynamic simulations based on X-ray structures of distinct B30.2 domain dimers to identify the asymmetric dimer as most active, which has implications for the inside-out signaling mechanism.

BACKGROUND Xinong Saanen goat milk is a raw material for goat milk-based infant formula production. This study aims to analyze digestion properties of Xinong Saanen goat colostrum and mature milk by simulating infant gastrointestinal digestion. Zeta potential, particles size, protein profile and peptides composition of these two kinds of milk during the digestion process were studied. RESULTS Zeta-potential values of the digested colostrum were lower than those of mature milk through the whole digestion. Absolute zeta potential of colostrum duodenal digestion samples showed a decrease from 16.63 ± 2.08 to 11.80 ± 2.03 mV while that of mature milk decreased sharply and then increased (P

Milk fat globules (MFGs), which are secreted by the epithelial cells of the lactating mammary glands, account for the most of the nutritional value of milk. They are enveloped by the milk fat globule membrane (MFGM), a complex structure consisting of three phospholipid membrane monolayers and containing various lipids. Depending on the origin of milk, specific proteins accounts for 5-70% of the MFGM mass. Proteome of MFGMs includes hundreds of proteins, with nine major components being adipophilin, butyrophilin, cluster of differentiation 36, fatty acid binding protein, lactadherin, mucin 1, mucin 15, tail-interacting protein 47 (TIP47), and xanthine oxidoreductase. Two of the MFGM components, adipophilin and TIP47, belong to the five-member perilipin family of lipid droplet proteins. Adipophilin is involved in the formation of cytoplasmic lipid droplets and secretion of MFGs. This protein is also related to the formation of other lipid droplets that exist in most cell types, playing an important role in the transport of lipids from ER to the surface of lipid droplets. TIP47 acts as a cytoplasmic sorting factor for mannose 6-phosphate receptors and is recruited to the MFGM. Therefore, both adipophilin and TIP47 are moonlighting proteins, each possessing several unrelated functions. This review focuses on the main functions and specific structural features of adipophilin and TIP47, analyzes similarities and differences of these proteins among different species, and describes these proteins in the context of other members of the perilipin family.Communicated by Ramaswamy H. Sarma.

Churning is a common method to separate milk fat globule membrane (MFGM) from milk fat globules. In this work, we qualitatively and relative quantitatively analyzed the MFGM components obtained by churning cream for different time using a label-free proteomic approach. The result showed that 794 MFGM proteins were identified in all groups, of which 51, 14, 60 and 44 unique proteins were contained after churning cream for 2 min, 4 min, 6 min and 8 min, respectively. The sample churned for 6 min possessed the largest portion of total and unique proteins compared with other three groups. The relative abundances of proteins butyrophilin and xanthine dehydrogenase/oxidase, proteins cluster of differentiation 36 and fatty acid binding protein had the same change tendency during the continuous agitation process. Some milk serum proteins also were detected in the buttermilk. The analysis of protein profiles of MFGM at different churning time had an important guidance for enriching a certain MFGM protein.

Recent observation that prenyl pyrophosphates bind the Ig superfamily protein butyrophilin 3A1 (BTN3A1) suggests that modifying BTN3A1 activates major γδ T-cell subset, Vγ2Vδ2 T cells. Studies also show that microbial phosphoantigen HMBPP is required for expansion, pulmonary response, effector functions and memory polarization of Vγ2Vδ2 T cells during infections. Broad repertoires of cytokines involve expansion, recall-like expansion and effector functions of Vγ2Vδ2 T cells after Mtb infection or vaccination. Finally, mechanistic studies in nonhuman primate TB model demonstrate early expansion and differentiation of Vγ2Vδ2 T cells during Mtb infection can increase immune resistance to TB in macaques, with a potential mechanism of early/sustained IFN-γ production and CTL killing.

Cancer Immunology T lymphocytes are immune cells that can be activated through their gamma delta (γδ) or alpha beta (αβ) receptors. Both T cell types are found in human cancers, but current immunotherapies do not harness their coordinated antitumor activity. Payne et al. found that BTN3A1 and BTN2A1, two members of the butyrophilin family of proteins, partner to activate the most abundant subset of γδ T cells in peripheral blood. Antibodies targeting BTN3A1 redirect γδ T cells to attack cancer cells while also increasing the activity of tumor-specific αβ T cells. Thus, the killing of established tumors by different T cell subsets can be achieved through BTN3A1 targeting and may provide new strategies for cancer immunotherapy. Science , this issue p. [942][1] [1]: /lookup/doi/10.1126/science.aay2767

Citrullination is a post-translational modification (PTM) in which positively charged peptidyl-arginine is converted into neutral peptidyl-citrulline by peptidylarginine deiminase (PAD or PADI) enzymes. The full protein citrullinome in many tissues is unknown. Herein, we used mass spectrometry and identified 107 citrullinated proteins in the lactation day 9 (L9) mouse mammary gland including histone H2A, &alpha, tubulin, and &beta, casein. Given the importance of prolactin to lactation, we next tested if it stimulates PAD-catalyzed citrullination using mouse mammary epithelial CID-9 cells. Stimulation of CID-9 cells with 5 &micro, g/mL prolactin for 10 min induced a 2-fold increase in histone H2A citrullination and a 4.5-fold increase in &alpha, tubulin citrullination. We next investigated if prolactin-induced citrullination regulates the expression of lactation genes &beta, casein (Csn2) and butyrophilin (Btn1a1). Prolactin treatment for 12 h increased &beta, casein and butyrophilin mRNA expression, however, this increase was significantly inhibited by the pan-PAD inhibitor, BB-Cl-amidine (BB-ClA). We also examined the effect of tubulin citrullination on the overall polymerization rate of microtubules. Our results show that citrullinated tubulin had a higher maximum overall polymerization rate. Our work suggests that protein citrullination is an important PTM that regulates gene expression and microtubule dynamics in mammary epithelial cells.

Immunology In contrast to the well-studied αβ T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) and MHC-like molecules, how γδ T cells recognize antigens remains largely a mystery. One major class of γδ T cells, designated Vγ9Vδ2+, is activated by small, phosphorylated nonpeptide antigens, or phosphoantigens, produced by microbes and cancer cells. Rigau et al. found that these cells needed the combination of two immunoglobulin superfamily members, butyrophilin 2A1 (BTN2A1) and BTN3A1, on their cell surface to recognize these phosphoantigens. BTN2A1 directly binds the Vγ9+ domain of the T cell receptor (TCR), whereas a second ligand, potentially BTN3A1, binds the Vδ2 and γ-chain regions on the opposite side of the TCR. A better understanding of this unexpected form of T cell antigen recognition should inform and enhance future γδ T cell–mediated immunotherapies. Science , this issue p. [eaay5516][1] [1]: /lookup/doi/10.1126/science.aay5516

Summary Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1’s P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner., Graphical Abstract, Highlights • Radiation hybrids identify BTN2A1 as crucial for Vγ9Vδ2 phosphoantigen (P-Ag) sensing • BTN2A1 binds directly to the T cell receptor via germline-encoded regions of Vγ9 • Cell-surface BTN2A1 associates directly with BTN3A1 independent of P-Ag stimulation • The Vγ9-BTN2A1 interaction modality suggests an additional CDR3-dependent TCR ligand, Karunakaran et al. find that butyrophilin 2A1 (BTN2A1) associates with BTN3A1 on the cell surface and binds directly to germline-encoded regions of the Vγ9 chain of the Vγ9Vδ2 TCR. Thus, BTN2A1 collaborates with BTN3A1 to potentiate Vγ9Vδ2 T cell recognition, playing an essential role in phosphoantigen sensing.

Small organophosphorus compounds stimulate Vγ9Vδ2 T cells if they serve as ligands of butyrophilin 3A1. Because the most potent natural ligand is (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), which is the last intermediate in bacterial biosynthesis of isoprenoids that is not found in mammalian metabolism, activation of these T cells represents an important component of the immune response to bacterial infections. To identify butyrophilin ligands that may have greater plasma stability, and clinical potential, we have prepared a set of aryl phosphonamidate derivatives (9a-i) of the natural ligand. Testing of these new compounds in assays of T cell response has revealed that this strategy can provide compounds with high potency for expansion of Vγ9Vδ2 T cells (9f, EC(50) = 340 pM) and interferon γ production in response to loaded K562 cells (9e, EC(50) = 62 nM). Importantly, all compounds of this class display extended plasma stability (t(1/2) > 24 h). These findings increase our understanding of metabolism of butyrophilin ligands and the structure-activity relationships of phosphonamidate prodrugs.

Despite playing critical roles in the immune response and having significant potential in immunotherapy, γδ T cells have garnered little of the limelight. One major reason for this paradox is that their antigen recognition mechanisms are largely unknown, limiting our understanding of their biology and our potential to modulate their activity. One of the best-studied γδ subsets is the human Vγ9Vδ2T cell population, which predominates in peripheral blood and can combat both microbial infections and cancers. Although it has been known for decades that Vγ9Vδ2T cells respond to the presence of small pyrophosphate-based metabolites, collectively named phosphoantigens (pAgs), derived from microbial sources or malignant cells, the molecular basis for this response has been unclear. A major breakthrough in this area came with the identification of the Butyrophilin 3A (BTN3A) proteins, members of the Butyrophilin/Butyrophilin-like protein family, as mediators between pAgs and Vγ9Vδ2T cells. In this article, we review the most recent studies regarding pAg activation of human Vγ9Vδ2T cells, mainly focusing on the role of BTN3A as the pAg sensing molecule, as well as its potential impact on downstream events of the activation process.

Butyrophilins (BTNs) are a group of the moonlighting proteins, some members of which are secreted in milk. They constitute a large family of structurally similar type 1 transmembrane proteins from the immunoglobulin superfamily. Although the founding member of this family is related to lactation, participating in the secretion, formation and stabilization of milk fat globules, it may also have a cell surface receptor function. Generally, the BTN family members are known to modulate co-stimulatory responses, T cell selection, differentiation, and cell fate determination. Polymorphism of these genes was shown to be associated with the pathology of several human diseases. Despite their biological significance, structural information on human butyrophilins is rather limited. Based on their remarkable multifunctionality, butyrophilins seem to belong to the category of moonlighting proteins, which are known to contain intrinsically disordered protein regions (IDPRs). However, the disorder status of human BTNs was not systematically investigated as of yet. The goal of this study is to fill this gap and to evaluate peculiarities of intrinsic disorder predisposition of the members of human BTN family, and to find if they have IDPRs that can be attributed to the multifunctionality of these important proteins.

In this study, bovine mammary epithelial cells were used to study stress responses after cells were exposed to 42°C for 0.5, 1, 3, 5, 8 or 12 h, and 38°C as control. The transcription of the genes (HSP27, HSP70 and HSP90) of heat shock protein (Hsp) was significantly enhanced under heat stress (HS). The peak transcription of HSP70 was 14 times the control at 1 h. Expression of proteins Hsp27 and Hsp70 was gradually increased under HS, with rapid deposition of Hsp70 in epithelial cells. The major milk protein genes of β-casein (CSN2) and butyrophilin (BTN1A1) were down-regulated and the synthesis of total caseins was decreased. After the cells were under HS (42°C) for 1 or 5 h, the cells were cultured at 38°C for 1, 6, 12 or 24 h for recovery. When the cells were cultured at 38°C for 24 h after HS for 1 h, the transcription of HSP70, HSP90, CSN2 and BTN reached normal levels. Our results suggest that HS initiated Hsp synthesis and decreased the milk protein synthesis. Hsp70 is extremely sensitive to HS and mainly responsible for mammary cell protection from HS.

Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 10(5) to 10(6) cells/mL were analyzed qualitatively and quantitatively using both one-dimension sodium dodecyl sulfate PAGE and filter-aided sample preparation coupled with dimethyl labeling, both followed by liquid chromatography tandem mass spectrometry. Minor differences were found on the qualitative level in the proteome from milk with different SCC levels, whereas the concentration of milk proteins showed remarkable changes. Not only immune-related proteins (cathelicidins, IGK protein, CD59 molecule, complement regulatory protein, lactadherin), but also proteins with other biological functions (e.g., lipid metabolism: platelet glycoprotein 4, butyrophilin subfamily 1 member A1, perilipin-2) were significantly different in milk from cows with high SCC level compared with low SCC level. The increased concentration of protease inhibitors in the milk with higher SCC levels may suggest a protective role in the mammary gland against protease activity. Prostaglandin-H2 D-isomerase showed a linear relation with SCC, which was confirmed with an ELISA. However, the correlation coefficient was lower in individual cows compared with bulk milk. These results indicate that prostaglandin-H2 D-isomerase may be used as an indicator to evaluate bulk milk quality and thereby reduce the economic loss in the dairy industry. The results from this study reflect the biological phenomena occurring during subclinical mastitis and in addition provide a potential indicator for the detection of bulk milk with high SCC.

Human Vγ9/Vδ2 T-cells detect tumour cells and microbial infections by recognising 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 are discriminated from non-antigenic small molecules. Here, we utilised 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 non-antigenic 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.

Human Vγ9Vδ2 T cells respond to microbial infections as well as certain types of tumors. The key initiators of Vγ9Vδ2 activation are small, pyrophosphate-containing molecules called phosphoantigens (pAgs) that are present in infected cells or accumulate intracellularly in certain tumor cells. Recent studies demonstrate that initiation of the Vγ9Vδ2 T cell response begins with sensing of pAg via the intracellular domain of the butyrophilin 3A1 (BTN3A1) molecule. However, it is unknown how downstream events can ultimately lead to T cell activation. Here, using NMR spectrometry and molecular dynamics (MD) simulations, we characterize a global conformational change in the B30.2 intracellular domain of BTN3A1 induced by pAg binding. We also reveal by crystallography two distinct dimer interfaces in the BTN3A1 full-length intracellular domain, which are stable in MD simulations. These interfaces lie in close proximity to the pAg-binding pocket and contain clusters of residues that experience major changes of chemical environment upon pAg binding. This suggests that pAg binding disrupts a preexisting conformation of the BTN3A1 intracellular domain. Using a combination of biochemical, structural, and cellular approaches we demonstrate that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminishes pAg-induced T cell activation. Based on these results, we propose a model in which a conformational change in BTN3A1 is a key event of pAg sensing that ultimately leads to T cell activation.

Comment onButyrophilin 3A (BTN3A, CD277)-specific antibody 20.1 differentially activates Vγ9Vδ2 TCR clonotypes and interferes with phosphoantigen activation. [Eur J Immunol. 2017]; International audience; Human blood γδ T lymphocytes express TCRVγ9Vδ2 and respond to nonpeptide phosphoantigens (PAgs) by a mysterious mechanism involving the BTN3A1 (CD277) molecule . BTN3A1 is a butyrophilin-like protein related to CD80, PD-L1, and MHC, and is either a presenting or a co-stimulatory molecule for PAgs. Although the precise roles and molecular interactions with the TCRVγ9Vδ2 are currently not determined, it is commonly thought that all TCRVγ9Vδ2 lymphocytes 'see' PAg and BTN3A1 together, presumably in a single molecular recognition event. But whether this recognition event could be reproduced in a simplified model was not addressed in previous studies. In this issue, Starick et al. (Eur. J. Immunol. 2017. 47: 982-992) compared the response of three TCRVγ9Vδ2 pairs of murine and human cell transfectants to PAg and anti-BTN3A1 antibodies using IL-2 release as a readout. The authors found that although the two murine transfectants responded similarly to either stimuli, one murine TCRVγ9Vδ2 transfectant reacted to PAgs but not to anti-BTN3A1 (mAb 20.1). Human transductants behave in a similar fashion, demonstrating that TCRVγ9Vδ2 lymphocytes differentiate PAg and BTN3A1 signals, while species of the transductants unmask this differential sensitivity. Indeed, understanding the puzzling mode of antigen recognition by γδ T lymphocytes will be essential for developing γδ T-cell-based immunotherapies, and the authors of this study now demonstrate that TCRVγ9Vδ2 lymphocytes are able to differentiate the PAg and BTN3A1 stimuli.

The butyrophilin 3 (BTN3) receptors are implicated in the T lymphocytes regulation and present a wide plasticity in mammals. In order to understand how these genes have been diversified, we studied their evolution and show that the three human BTN3 are the result of two successive duplications in Primates and that the three genes are present in Hominoids and the Old World Monkey groups. A thorough phylogenetic analysis reveals a concerted evolution of BTN3 characterized by a strong and recurrent homogenization of the region encoding the signal peptide and the immunoglobulin variable (IgV) domain in Hominoids, where the sequences of BTN3A1 or BTN3A3 are replaced by BTN3A2 sequence. In human, the analysis of the diversity of these genes in 1683 individuals representing 26 worldwide populations shows that the three genes are polymorphic, with more than 46 alleles for each gene, and marked by extreme homogenization of the IgV sequences. The same analysis performed for the BTN2 genes shows also a concerted evolution; however, it is not as strong and recurrent as for BTN3. This study shows that BTN3 receptors are marked by extreme concerted evolution at the IgV domain and that BTN3A2 plays a central role in this evolution.

We describe the case of a boy with acute myeloid leukemia with translocation t(6;11)(p22.2;q23) and insertion ins(11;9)(q23;p21.3p21.3). Translocation t(6;11)(p22.2;q23) involving the short arm of chromosome 6 has not been previously described. The LDI-PCR showed the presence of KMT2A-MLLT3 fusion and identified the BTN3A1 (butyrophilin subfamily 3 member A1) gene on 6p22.2 as the other KMT2A translocation partner. The BTN3A1 gene has never been described in the context of acute leukemia. Although this fusion is out of frame, as the antisense strand of BTN3A1 is fused to the sense strand of KMT2A, the loss of heterozygosity of the BTN3A1 gene might contribute to the malignancy of leukemic cells.

SummaryPhosphoantigen-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.

Pyrophosphorylated metabolites of isoprenoid-biosynthesis (phosphoantigens, PAgs) activate Vγ9Vδ2 T cells during infections and trigger antitumor activity. This activation depends on expression of butyrophilin 3 A1 (BTN3A1) by antigen-presenting cells. This report defines the minimal genetic requirements for activation of Vγ9Vδ2 T cells by PAgs and mAb 20.1. We compared PAg-presentation by BTN3A1-transduced CHO hamster cells with that of CHO cells containing the complete human chromosome 6 (Chr6). BTN3A1 expression alone was sufficient for activation of Vγ9Vδ2 T-cell receptor transductants by mAb 20.1., while activation by PAgs also required the presence of Chr6. We take this finding as evidence that gene(s) on Chr6 in addition to BTN3A1 are mandatory for PAg-mediated activation of Vγ9Vδ2 T cells. This observation is important for the design of animal models for PAg-mediated immune responses and provokes speculations about the analogy between genes controlling PAg presentation and MHC-localized genes controlling peptide-antigen presentation.

Summary In this study, changes in gene effects for milk production traits were analysed over time. Such changes can be expected by investigating daily milk production yields, which increase during the early phase of lactation and then decrease. Moreover, additive polygenic effects on milk production traits estimated in other studies differed throughout the 305 days of lactation, clearly indicating changes in the genetic determination of milk production throughout this period. Our study focused on particular candidate genes known to affect milk production traits and on the estimation of potential changes in the magnitude of their effects over time. With two independent data sets from Holstein-Friesian and Jersey breeds, we show that the effects of the DGAT1:p.Lys232Ala polymorphism on fat and protein content in milk change during lactation. The other candidate genes considered in this study (leptin receptor, leptin and butyrophilin, subfamily 1, member A1) exhibited effects that vary across time, but these could be observed in only one of the breeds. Longitudinal modelling of SNP effects enables more precise description of the genetic background underlying the variation of complex traits. A gene that changes the magnitude or even the sign of its effect cannot be detected by a time-averaged model. This was particularly evident when analysing the effect of butyrophilin, missed by many previous studies, which considered butyrophilin's effect as constant over time.

The impact of polymorphism of the diacylglycerol acyltransferase ( DGAT1 ), butyrophilin ( BTN1A1 ), oxidized low-density lipoprotein receptor ( OLR1 ), and signal transducer and activator of transcription 1 ( STAT1 ) genes on milk production and reproduction traits in 419 Czech Fleckvieh cows was examined using polymerase chain reaction and restriction fragment length polymorphism. The loci DGAT1 and BTN1A1 were observed simultaneously to affect milk production, estimated breeding value of milk production traits, as well as repro-duction parameters. Significant differences were found also between genotypes of the STAT1 loci in relation to estimated breeding value of milk production traits. Similar findings in pure dairy breeds suggest that heterogene -ous effects of the observed loci can be explained by different genetic backgrounds in various breed populations selected to achieve different commercial goals. Thus, it is necessary to determine variability and influence of a molecular marker in a specific population when considering its inclusion into a breeding programme.

Human Vγ9Vδ2 T cells recognize phosphorylated products of isoprenoid metabolism (phosphoantigens) PAg with TCR comprising Vγ9JP γ-chains and Vδ2 δ-chains dependent on butyrophilin 3 (BTN3) expressed by antigen-presenting cells. They are massively activated in many infections and show anti-tumor activity and so far, they have been considered to exist only in higher primates. We performed a comprehensive analysis of databases and identified the three genes in species of both placental magnorders, but not in rodents. The common occurrence or loss of in silico translatable Vγ9, Vδ2, and BTN3 genes suggested their co-evolution based on a functional relationship. In the peripheral lymphocytes of alpaca (Vicugna pacos), characteristic Vγ9JP rearrangements and in-frame Vδ2 rearrangements were found and could be co-expressed in a TCR-negative mouse T cell hybridoma where they rescued CD3 expression and function. Finally, database sequence analysis of the extracellular domain of alpaca BTN3 revealed complete conservation of proposed PAg binding residues of human BTN3A1. In summary, we show emergence and preservation of Vγ9 and Vδ2 TCR genes with the gene of the putative antigen-presenting molecule BTN3 in placental mammals and lay the ground for analysis of alpaca as candidate for a first non-primate species to possess Vγ9Vδ2 T cells.