Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5.

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

Evaluating Natural Language Processing Packages for Predicting Hospital-Acquired Pressure Injuries From Clinical Notes.

Incidence of hospital-acquired pressure injury, a key indicator of nursing quality, is directly proportional to adverse outcomes, increased hospital stays, and economic burdens on patients, caregivers, and society. Thus, predicting hospital-acquired pressure injury is important. Prediction models use structured data more often than unstructured notes, although the latter often contain useful patient information. We hypothesize that unstructured notes, such as nursing notes, can predict hospital-acquired pressure injury. We evaluate the impact of using various natural language processing packages to identify salient patient information from unstructured text. We use named entity recognition to identify keywords, which comprise the feature space of our classifier for hospital-acquired pressure injury prediction. We compare scispaCy and Stanza, two different named entity recognition models, using unstructured notes in Medical Information Mart for Intensive Care III, a publicly available ICU data set. To assess the impact of vocabulary size reduction, we compare the use of all clinical notes with only nursing notes. Our results suggest that named entity recognition extraction using nursing notes can yield accurate models. Moreover, the extracted keywords play a significant role in the prediction of hospital-acquired pressure injury.

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics.

CC chemokine receptor 5 (CCR5) contributes to inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A drug against CCR5 has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of inflammatory diseases and cancer has had limited success because of the complex biology and pharmacology of this receptor. CCR5 is activated by many natural and engineered chemokines that elicit distinct receptor signaling and trafficking responses, including some that sequester the receptor inside the cell. The sequestration phenomenon may be therapeutically exploitable, but the mechanisms by which different ligands traffic CCR5 to different cellular locations are poorly understood. Here we employed live cell ascorbic acid peroxidase proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveals distinct ligand-dependent CCR5 trafficking patterns with temporal resolution. All three chemokines internalize CCR5 via ß-arrestin- dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and with varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the molecular mechanisms behind CCR5 intracellular sequestration and suggest actionable patterns for the development of chemokine-based CCR5 targeting molecules. Significance Statement: CCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and HIV transmission. Along with its functional diversity, different CCR5 ligands can induce distinct receptor signaling responses and trafficking behaviors; the latter includes intracellular receptor sequestration which offers a potential therapeutic strategy for inhibiting CCR5 function. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the molecular basis for receptor sequestration including information that can be exploited for the development of CCR5 targeting molecules that promote retention of the receptor inside the cell.

On-bead purification and nanodisc reconstitution of human chemokine receptor complexes for structural and biophysical studies.

Chemokine receptors, a subfamily of G-protein-coupled receptors (GPCRs), are responsible for cell migration during physiological processes as well as in diseases like inflammation and cancers. Here, we present a protocol for solubilizing, purifying, and reconstituting complexes of chemokine receptors with their ligands in "nanodiscs," soluble lipid bilayers that mimic the native environment of membrane receptors. The protocol yields chemokine receptor complexes with sufficient purity and yield for structural and biophysical studies and should be applicable to other GPCRs.

An injectable and active hydrogel induces mutually enhanced mild magnetic hyperthermia and ferroptosis.

Magnetic hyperthermia therapy (MHT) is a promising new modality to deal with solid tumors, yet the low magnetic-heat conversion efficacy, magnetic resonance imaging (MRI) artifacts, easy leakage of magnetic nanoparticles, and thermal resistance are the main obstacles to expand its clinical applications. Herein, a synergistic strategy based on a novel injectable magnetic and ferroptotic hydrogel is proposed to overcome these bottlenecks and boost the antitumor efficacy of MHT. The injectable hydrogel (AAGel) exhibiting a sol-gel transition upon heating is made of arachidonic acid (AA)-modified amphiphilic copolymers. Ferrimagnetic Zn0.4Fe2.6O4 nanocubes with high-efficiency hysteresis loss mechanism are synthesized and co-loaded into AAGel with RSL3, a potent ferroptotic inducer. This system maintains the temperature-responsive sol-gel transition, and provides the capacity of multiple MHT and achieves accurate heating after a single injection owing to the firm anchoring and uniform dispersion of nanocubes in the gel matrix. The high magnetic-heat conversion efficacy of nanocubes coupled with the application of echo limiting effect avoids the MRI artifacts during MHT. Besides the function of magnetic heating, Zn0.4Fe2.6O4 nanocubes combined with multiple MHT can sustain supply of redox-active iron to generate reactive oxygen species and lipid peroxides and accelerate the release of RLS3 from AAGel, thus enhancing the antitumor efficacy of ferroptosis. In turn, the reinforced ferroptosis can alleviate the MHT-triggered thermal resistance of tumors by impairment of the protective heat shock protein 70. The synergy strategy achieves the complete elimination of CT-26 tumors in mice without causing local tumor recurrence and other severe side effects.

Accuracy of newer generation intraocular lens power calculation formulas in pediatric cataract patients.

PURPOSE: To evaluate the accuracy of newer generation intraocular lens (IOL) power calculation formulas (EVO 2.0 and Kane) with established formulas (Barrett Universal II, Haigis and SRK/T) in pediatric cataract patients. METHODS: Retrospective study. We enrolled 110 eyes (110 patients) in Eye Hospital of Wenzhou Medical University. All patients underwent uneventful cataract surgery and implanted with posterior chamber IOL in the bag. We calculate the mean prediction errors (PE) and percentage within 1 diopter (D) at 1 month to assess the accuracy, and percentage > 2D was defined as prediction accident. Then, we performed subgroup analysis according to age and axial length (AL). RESULTS: The mean age and AL were 37.45 ± 23.28 months and 21.16 ± 1.29 mm. The mean PE for all patients was as follows: Barrett (- 0.30), EVO (0.18), Haigis (- 0.74), Kane (- 0.36), and SRK/T (0.58), p < 0.001. In addition, EVO and SRK/T formulas were relatively accurate in patients younger than 24 months and with AL ≤ 21 mm, while EVO got lower prediction accident rate than SRK/T (3/41 vs 8/41, 4/52 vs 5/52). Moreover, Barrett, EVO, and Kane formulas achieved better accuracy and lower prediction accident rate in patients older than 24 months and with AL > 21 mm (both > 51/69 and 43/58, and < 3/69 and 3/58). CONCLUSIONS: In patients older than 24 months and with AL > 21 mm, Barrett, EVO, and Kane formulas were relatively accurate, while in patients younger than 24 months and with AL ≤ 21 mm, EVO was more accurate, followed by SRK/T formula.

Investigate the multipath erasure of nitrobenzene over nanoscale zero-valent-iron/N-doped biochar hybrid with extraordinary reduction performance.

In this study, the facile carbothermal reduction method was enforced using urea as dopant to modify the structure and chemical composition of nanoscale zero-valent-iron/biochar hybrid thereby boosting its reduction performance. Through fine-tuning the N-doped amount, the optimal nZVI/N-doped BC was obtained, which exhibited more active sites (nZVI, persistent free radicals (PFRs), pyrrolic-N) and superior electrochemical conductivity. With these blessings, the electrons originating from galvanic cell reaction could zip along the highway within the hybrid. Taking nitrobenzene (NB) as the target pollutant, the quantitative analysis revealed that the NB reduction and adsorption removal efficiency were dramatically improved by 2.42 and 2.78 times, respectively. What's more, combining the in-situ experimental detection and theoretical calculations, unexpected NB reductive multipath with respect to PFRs and pyrrolic-N accelerating the Fe3+/Fe2+ cycle within the nZVI/N-doped BC system was decoded. The enhancement of Fe3+/Fe2+ cycle improved the electron utilization efficiency and maintained the reduction reactivity of the hybrid. This work raised awareness of the mechanisms regarding the reduction performance of nZVI/N-doped BC elevated by N-doped and the pollutant reductive pathway within the system, uncovered the dusty roles of PFRs and N-species during the reduction process.

A Retrospective Study on the Eye-Related Quality of Life, Functional Vision, and Their Determinants Among Children Following Congenital and Developmental Cataracts Surgery and Its Impact on Their Families Using the PedEyeQ.

Objectives: To evaluate the eye-related quality of life (ER-QOL), functional vision, and their determinants in children following congenital and developmental cataract surgery, as the impact on their families, using the Pediatric Eye Questionnaire (PedEyeQ). Materials and Methods: This was a retrospective cross-sectional study involving 107 children (aged 0-11 years) with congenital and developmental cataracts who had undergone surgery, as well as 59 visually healthy controls (aged 0-11 years). One parent for each child completed either the Proxy 0-4 PedEyeQ, the Proxy 5-11 PedEyeQ, or the Parent PedEyeQ, depending on their child's age. Mann-Whitney U and Kruskal-Wallis tests were then conducted to compare the differences between groups and to analyze their determinants. Results: PedEyeQ scores were found to be lower in postoperative children with congenital and developmental cataracts compared with the control group across all study domains. The PedEyeQ Proxy 0 to 4 years' greatest mean difference was 27 points worse in the Functional Vision domain (95% CI -34 to -19; p < 0.001). We also found that the occurrence of nystagmus (p < 0.005) and strabismus (p < 0.005) were the major factors affecting participants' functional vision. The PedEyeQ Proxy 5 to 11 years' greatest mean difference was 23 points worse in this same domain (95% CI -30 to -15; p < 0.001), with nystagmus (p < 0.05) being the main determinant herein. Parent PedEyeQ 0 to 4 years' greatest difference was 46 points worse on the Worry about their Child's Eye Condition domain (95% CI -57 to -36; p < 0.001). Similarly, parents of children with ophthalmologic abnormalities, including nystagmus (p < 0.001) and strabismus (p < 0.05), were significantly more worried about their children's eye condition. Parent PedEyeQ 5 to 11 years' greatest difference was also found to be 30 points worse on the Worry about their Child's Eye Condition domain (95% CI -43 to -17; p < 0.005). Conclusions: Children who have undergone congenital and developmental cataract surgery experience a lower quality of life and reduced functional vision. Their families are also significantly and adversely affected herein. Thus, more attention is needed on these groups, with more focused measures being administered to both children and their families.

New insights into iron/nickel-carbon ternary micro-electrolysis toward 4-nitrochlorobenzene removal: Enhancing reduction and unveiling removal mechanisms.

The ternary micro-electrolysis material iron/nickel-carbon (Fe/Ni-AC) with enhanced reducibility was constructed by introducing the trace transition metal Ni based on the iron/carbon (Fe/AC) system and used for the removal of 4-nitrochlorobenzene (4-NCB) in solution. The composition and structures of the Fe/Ni-AC were analyzed by various characterizations to estimate its feasibility as reductants for pollutants. The removal efficiency of 4-NCB by Fe/Ni-AC was considerably greater than that of Fe/AC and iron/nickel (Fe/Ni) binary systems. This was mainly due to the enhanced reducibility of 4-NCB by the synergism between anode and double-cathode in the ternary micro-electrolysis system (MES). In the Fe/Ni-AC ternary MES, zero-iron (Fe0) served as anode involved in the formation of galvanic couples with activated carbon (AC) and zero-nickel (Ni0), respectively, where AC and Ni0 functioned as double-cathode, thereby promoting the electron transfer and the corrosion of Fe0. The cathodic and catalytic effects of Ni0 that existed simultaneously could not only facilitate the corrosion of Fe0 but also catalyze H2 to form active hydrogen (H*), which was responsible for 4-NCB transformation. Besides, AC acted as a supporter which could offer the reaction interface for in-situ reduction, and at the same time provide interconnection space for electrons and H2 to transfer from Fe0 to the surface of Ni0. The results suggest that a double-cathode of Ni0 and AC could drive much more electrons, Fe2+ and H*, thus serving as effective reductants for 4-NCB reduction.

Accuracy of Newer Generation IOL Power Calculation Formulas in Eyes With High Axial Myopia.

PURPOSE: To compare the accuracy of the Barrett Universal II, Emmetropia Verifying Optical (EVO), Haigis, Kane, and SRK/T formulas for intraocular lens power calculation in patients with high axial myopia. METHODS: In this retrospective study, 175 eyes (175 patients) that underwent uneventful cataract surgery were enrolled. According to the axial length (AL), the eyes were divided into long AL (26 ⩽ AL < 28 mm), super long AL (28 ⩽ AL < 30 mm), and extremely long AL (⩾ 30 mm). The mean absolute prediction errors (MAE) 3 months postoperatively and the percentage of eyes within different prediction error were compared, followed by subgroup analysis. RESULTS: The MAE and percentage of eyes within ±0.50 diopters (D) of the five formulas were as follows: Barrett Universal II (0.342, 74.9%), EVO 2.0 (0.314, 82.3%), Haigis (0.336, 74.9%), Kane (0.318, 78.9%), and SRK/T (0.398, 69.7%) (P = .552 and .071, respectively). Although no significant difference was found among the five formulas in the super and extremely long AL groups (P = .792 and .227, respectively), the EVO 2.0 formula achieved the highest accuracy (88.9%, 72 of 81) in the long AL group (P = .049). Moreover, the accuracy of the EVO 2.0 and Haigis formulas was stable, regardless of AL. The SRK/T formula showed a negative trend in the long and super long AL groups, whereas the Barrett Universal II, Kane, and SRK/T formulas showed positive trends in the extremely long AL group. CONCLUSIONS: Overall, the EVO 2.0 and Kane formulas achieved better results in patients with high axial myopia, whereas the other three formulas showed slightly poor outcomes. [J Refract Surg. 2021;37(11):754-758.].

B7-H3 on breast cancer cell MCF7 inhibits IFN-γ release from tumour-infiltrating T cells.

B7-H3 is a type I membrane protein that has contradictory co-stimulatory and co-inhibitory effects in adaptive and anti-tumour immunity. B7-H3 is up-regulated in many malignant tumours, including breast cancer. Therefore, we hypothesise that B7-H3, which has an immunosuppressive role, suppresses anti-tumour immunity. The aim of this study was to clarify the role of B7-H3 in the tumor microenvironment in breast cancer, explore the possibility of B7-H3 as a target for clinical immunotherapy, and provide reference for clinical work. We knocked down B7-H3 with siRNA in MCF7 breast cancer cells, which we termed MCF7-B7-H3-KD cells, and the expression of B7-H3 was assessed by flow cytometry. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) knockdown was used as a control (MCF7-Gapdh). MCF7-B7-H3-KD and MCF7-Gapdh cells were co-cultured with peripheral blood mononuclear cells (PBMCs) and CD3+ T cells from healthy donors to assess the effect of B7-H3 loss. PBMCs cultured with MCF7-Gapdh cells showed decreased activation, proliferation, and function of CD8+ T cells, but there was no effect on the proliferation of CD4+ T cells. However, when MCF7-B7-H3-KD cells were co-cultured with PBMCs, the proliferation ability of CD4+ T cells and CD8+ T cells was significantly higher than that observed in MCF7-Gapdh cell co-culture. Additionally, co-culture with MCF7-Gapdh cells decreased the expression of IFN-γ (Interferon-γ). However, after co-culture with MCF7-B7-H3-KD cells, there was an increase in IFN-γ. We further found that this inhibitory effect on IFN-γ was because of decreased mTOR (the mammalian target of rapamycin) phosphorylation in T cells. Treatment of T cells co-cultured with MCF7-B7-H3-KD cells with an mTOR inhibitor blocked the secretion of IFN-γ. B7-H3 on tumour cells inhibits the proliferation of CD4+ and CD8+ T cells and inhibits the release of IFN-γ by decreasing mTOR signalling. A better understanding of these complex immune regulatory mechanisms should facilitate the generation of more powerful and selective tools to manipulate cancer therapy.

Preoperative Management of MGD with Vectored Thermal Pulsation before Cataract Surgery: A Prospective, Controlled Clinical Trial.

Purpose: To investigate the efficacy of preoperative monocular treatment in elderly cataract patients with Meibomian Gland Dysfunction (MGD) utilizing vectored thermal pulsation treatment.Materials and Methods: This study was a prospective, examiner-masked contralateral eye clinical trial. Patients previously diagnosed with MGD undergoing uncomplicated cataract surgery in two eyes were enrolled. The eye perceived by the patient to be more symptomatic of MGD received a 12 min vectored thermal pulsation treatment using the LipiFlow Thermal Pulsation System, and was referred to as the LipiFlow-surgery eye. The contralateral eye then served as the nonLipiFlow-surgery eye. Patients with MGD not undergoing cataract surgery were enrolled as the control group. Within the control group, the eye that received LipiFlow treatment was considered the LipiFlow-nonsurgery eye, while the contralateral eye served as the nonLipiFlow-nonsurgery eye. All patients were examined before treatment and at one-week, one-month, and three-month intervals after treatment. Clinical parameters included dry eye symptoms, average lipid layer thickness (LLT-ave), tear breakup time (TBUT), corneal staining, Schirmer I tests, Meibomian glands yielding liquid secretion (MGYLS), and meibomian gland dropout.Results: A total of 32 patients (64 eyes) were examined during the three-month follow-up. There was a significant reduction in dry eye symptoms in non-surgery patients with monocular treatment of MGD, while no change in surgery patients was observed. Significant improvement of MGYLS in LipiFlow-surgery and LipiFlow-nonsurgery eyes during the follow-up time (p < .001) was reported, while no difference was observed in nonLipiFlow-surgery and nonLipiFlow-nonsurgery eyes. A statistically significant difference was seen in TBUT between LipiFlow-surgery and nonLipiFlow-surgery eyes at one-week and one-month intervals (p = .019 and 0.019, respectively). Differences in other clinical parameters were not statistically significant.Conclusions: Our findings suggest that although subjective symptoms were not alleviated, a single application of LipiFlow treatment before cataract surgery is effective in alleviating blockage of meibomian glands and preventing the decline of TBUT after cataract surgery.

Optimal fermentation time for Nigrospora-fermented tea rich in bostrycin.

BACKGROUND: Bostrycin has many biological functions, such as anticancer activity, and is becoming increasingly popular. Nigrospora sphaerica HCH285, which has the ability to produce high levels of bostrycin, can be used to ferment sun-dried green tea of Camellia sinensis through acclimation, resulting in the development of a Nigrospora-fermented tea. The effects of fermentation time on the production of bostrycin by the HCH285 strain were investigated. RESULTS: After 45 days of fermentation, the bostrycin content reached 3.18 g kg-1 , which is the highest level during the whole fermentation. At 50 days, the tea liquor was red, had a strong mushroom odour and a sweet taste, and presented optimal quality. The contents of free amino acids, tea polyphenols and soluble sugars in the fermented tea decreased generally during the fermentation, although the content of water-soluble substances increased. Additionally, the results of a 14-day acute oral toxicity test showed that Nigrospora-fermented tea was nontoxic. CONCLUSION: The optimum fermentation time of Nigrospora-fermented tea was concluded to be 45-50 days. These results provide insights with respect to the development of tea biotechnology and new tea products with active ingredients. © 2020 Society of Chemical Industry.

Synthesis of char-based adsorbents from cotton textile waste assisted by iron salts at low pyrolysis temperature for Cr(VI) removal.

Char-based adsorbents (char-FeCl3, char-FeCl2, and char-FeCit) derived from cotton textile waste (CTW) were synthesized by one-step low-temperature pyrolysis approach with different iron salts. The properties of the samples were conducted by BET, SEM, EDS, XRD, XPS, TEM, and FTIR. The results suggested that the surface areas of char-FeCl3 and char-FeCl2 were higher than those of char-FeCit. The presence of Fe2O3 as well as pyrolysis gas (HCl (g) and H2O (g)) could catalyze the formation of porosity. Meanwhile, FeCl3 showed the strongest catalysis effect to decompose cellulose to produce char. The pyrolysis process analysis was investigated by means of thermogravimetry-DSC. FeCl3 and FeCl2 could accelerate the breakage of cellulose structure whereas FeC6H5O7 was not beneficial to form char at low temperature as the incomplete decomposition of citrate. The adsorption property of Cr(VI) for the chars was evaluated. Adsorption processes were fitted well with the Freundlich model, and char-FeCl3 presented the best adsorptive capacity (70.39 mg/g). Thus, this low-temperature pyrolysis method was economical and technologically simplified as well as efficient adsorption capacity of Cr(VI) removal. Graphical abstract.

Butyrophilin3A proteins and Vγ9Vδ2 T cell activation.

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.

Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vγ9Vδ2 T cell activation.

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.

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.

RhoB Mediates Phosphoantigen Recognition by Vγ9Vδ2 T Cell Receptor.

Human Vγ9Vδ2 T cells respond to tumor cells by sensing elevated levels of phosphorylated intermediates of the dysregulated mevalonate pathway, which is translated into activating signals by the ubiquitously expressed butyrophilin A1 (BTN3A1) through yet unknown mechanisms. Here, we developed an unbiased, genome-wide screening method that identified RhoB as a critical mediator of Vγ9Vδ2 TCR activation in tumor cells. Our results show that Vγ9Vδ2 TCR activation is modulated by the GTPase activity of RhoB and its redistribution to BTN3A1. This is associated with cytoskeletal changes that directly stabilize BTN3A1 in the membrane, and the subsequent dissociation of RhoB from BTN3A1. Furthermore, phosphoantigen accumulation induces a conformational change in BTN3A1, rendering its extracellular domains recognizable by Vγ9Vδ2 TCRs. These complementary events provide further evidence for inside-out signaling as an essential step in the recognition of tumor cells by a Vγ9Vδ2 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.