Dialysis-related amyloidosis (DRA) is characterized by accumulation of amyloid β2- microglobulin (β2m) in the interstitial matrix. Matrix substances such as heparin have reportedly been strongly implicated in the pathogenesis of dialysis-related amyloidosis. In clinical setting of hemodialysis, two types of heparin, i.e., high and low molecular heparin (H.M.H. and L.M.H.) have been routinely used. Still commonly used is H.M.H., followed by L.M.H. preparations with distinct advantages. Here, we studied that the interaction of native and two amyloidogenic β2m variants: ΔN6β2m and D76N β2m with H.M.H. and L.M.H. We also investigated whether heparin could induce β2m to have an amyloidogenic conformation. Biolayer interferometry revealed that ΔN6β2m had a strong reaction and D76N β2m had a moderate reaction with H.M.H.. Furthermore,H.M.H. induced the C-terminal unfolding in a native β2m. By contrast, L.M.H. showed no reaction even with ΔN6β2m. This study showed firstly a direct binding of β2m with H.M.H.. H.M.H. would provoked a C-terminal unfolding of β2m, which indicated production of an amyloidogenic intermediate, i.e., β2m92-99. In addition, our findings also suggest that L.M.H. may provide beneficial effects against the development of the DRA. Since carpal tunnel syndrome was first reported in hemodialysis (HD) patients, why connective tissue was so often involved in dialysis-related amyloidosis (DRA) has been of particular interest in the investigation of the pathogenesis of this amyloidosis -5. Connein vitro [On the other hand, as is also well known, β2-microglobulin (β2m) is a precursor protein in DRA . In 1997in vitro . SubsequFurthermore, we determined that the C terminus from 92Ile to 99Met unfolded completely in an amyloidogenic variant, i.e., ΔN6β2m, which lacked the six N-terminal amino acids .Recently, Valleix et al. reported the first naturally occurring structural variant of β2m, Asp76Asn β2m (D76N β2m), discovered in members of a French family who developed progressive bowel dysfunction with extensive visceral β2m amyloid deposits despite normal renal function and normal circulating β2m concentrations and with none of the osteoarticular deposits characteristic of dialysis-related amyloidosis .In this study, therefore, we analyzed the binding of native β2m with heparin and compared it with that of two amyloidogenic β2m variants: ΔN6β2m and D76N β2m. We then investigated whether heparin at a clinical dosage could induce C-terminal unfolding. We also suggested that serum β2m concentrations of 2.0 μM might be set as the target level before HD  in HD patients.β2m and heparin: Purified β2m, which was used as the native β2m, and two kinds of heparin, i.e., H.M.H., and L.M.H.(4000-6000 M.W.) were purchased from Sigma .ΔN6β2m and Asp76Asn β2m (D76N β2m): As previously reported, ΔN6β2m and Asp76Asn β2m (D76N β2m) were produced at the genetic engineering laboratory of Hokkaido System Science Co., Ltd.  [, Japan) .mAb92-99: A monoclonal antibody specific for the C terminus of β2m (mAb92-99) was produced, as reported earlier [Biolayer interferometry (BLI) analysis: BLI binding experiments were conducted at room temperature with a BLItz instrument  [CA, USA) . BrieflyBinding of the native β2m, ΔN6β2m, and D76N β2m with heparin was studied at 0.1 and 1.0 μM by means of BLI. ΔN6β2m showed a strong reaction and D76N β2m showed a moderate reaction with H.M.H. . The BLIL.M.H. showed no reaction even with ΔN6β2m .We next investigate whether the C terminus of D76N β2m unfolded. BLI at 0.1 μM D76N β2m clearly showed binding with mAb92-99, which indicated the C-terminal unfolding of D76N β2m . The Kd in vivo [Recent basic studies demonstrated that SO3− groups associated with GAGs are strongly implicated in β2m amyloidogenesis and that the interstitial matrix is a site that contains amyloidogenic β2m, which contributes to development of DRA in vivo , 14. Borin vivo . ΔN6β2m in vivo , 17. TheBiotinylated H.M.H. (0.5 U/mL) was immobilized onto streptavidin biosensor. Protein concentrations were 1.0 µM (A) and 0.1 µM (B), respectively. ΔN6β2m , D76N β2m  and native β2m . (C) Biotinylated H.M.H. (0.5 U/mL) was immobilized onto streptavidin biosensor and native β2m  was used as the binding partner. (D) Biotinylated L.M.H. (5.0 U/mL) was immobilized onto streptavidin biosensor. ΔN6β2m  and native β2m  were used as the binding partner. Protein concentration was 4.0 µM. Three- independent experiments were performed, respectively. mAb92-99 (10 µg/mL) was immobilized onto protein A biosensor and D76N β2m (1.0 µM) was used a as the binding partner (A). After adding native β2m (1.0 µM) to the reaction drop holder in the presence or absence of H.M.H. (0.5 U/mL), the real-time binding was monitored. Native β2m incubated with H.M.H and L.M.H. for 24 h was also used as the binding partner (B). Three-independent experiments were performed.We previously reported that an amyloidogenic intermediate β2m, i.e., β2m92-99, similar to ΔN6β2m, may also be formed via interactions with GAGs in the extravascular space .This study not only confirmed the binding of two amyloidogenic β2m variants, i.e., ΔN6β2m and D76N β2m, with H.M.H. but also suggested the binding potential of native β2m with H.M.H. . In addiWhereas, a dose-dependent study with native β2m that was incubated with H.M.H. showed the BLI response value to be equal to 0.2 nm at 2.0 μM . Given tin vivo concentrations of GAGs with SO3− groups in HD patients, but the heparin concentration of 0.5 unit/ml used in this study may be comparable to serum concentrations in HD patients undergoing systemic heparinization. Relini et al. [We are not certain about the true i et al.  previousi et al. . Bonysiki et al.  showed tHeparin has been implicated also in other amyloidosis—in Alzheimer’s disease and systemic amyloidosis associated with serum amyloid A protein . Ariga eFinally, this study might demonstrated definitely clinical advantage of L.M.H. compared with H.M.H. for prevention of development of DRA. An underlying mechanism by which L.M.H. give rise to less interaction with β2m might be due to few contents of SO3− group.


SL mice (harboring the Swedish and London mutation in human amyloid precursor protein (APP)) which are characterized by enhanced AβPP processing and cerebral mitochondrial dysfunction, representing a mouse model of early AD.Current approved drugs for Alzheimer’s disease (AD) only attenuate symptoms, but do not cure the disease. The pirinixic acid derivate MH84 has been characterized as a dual gamma-secretase/proliferator activated receptor gamma (PPARγ) modulator in vitro. Pharmacokinetic studies in mice showed that MH84 is bioavailable after oral administration and reaches the brain. We recently demonstrated that MH84 improved mitochondrial dysfunction in a cellular model of AD. In the present study, we extended the pharmacological characterization of MH84 to 3-month-old Thy-1 AβPPSL mice received 12 mg/kg b.w. MH84 by oral gavage once a day for 21 days. Mitochondrial respiration was analyzed in isolated brain mitochondria, and mitochondrial membrane potential and ATP levels were determined in dissociated brain cells. Citrate synthase (CS) activity was determined in brain tissues and MitoTracker Green fluorescence was measured in HEK293-AβPPwt and HEK293-AβPPsw cells. Soluble Aβ1–40 and Aβ1–42 levels were determined using ELISA. Western blot analysis and qRT-PCR were used to measure protein and mRNA levels, respectively.Three-month-old Thy-1 AβPPMH84 reduced cerebral levels of the β-secretase-related C99 peptide and of Aβ40 levels. Mitochondrial dysfunction was ameliorated by restoring complex IV (cytochrome-c oxidase) respiration, mitochondrial membrane potential, and levels of ATP. Induction of PPARγ coactivator-1α (PGC-1α) mRNA and protein expression was identified as a possible mode of action that leads to increased mitochondrial mass as indicated by enhanced CS activity, OXPHOS levels, and MitoTracker Green fluorescence.MH84 modulates β-secretase processing of APP and improves mitochondrial dysfunction by a PGC-1α-dependent mechanism. Thus, MH84 seems to be a new promising therapeutic agent with approved in-vivo activity for the treatment of AD.The online version of this article (10.1186/s13195-018-0342-6) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is an age-related neurodegenerative disease and the primary cause for dementia in the elderly which represents a growing public health issue . After aBeside its effects on AβPP processing and Aβ-mediated cell death, including reduced BACE1 expression and transcription, decreased BACE1 promoter activity, and a clearance mechanism for Aβ –19, PPARPeroxisome proliferator-activated receptor-gamma coactivator alpha (PGC-1α) is a nuclear factor that regulates mitochondrial biogenesis in response to diverse environmental stimuli . PGC-1α SL mice which are characterized by enhanced AβPP processing and cerebral mitochondrial dysfunction, representing a mouse model of early AD [The pirinixic adic derivate MH84 -phenethoxy)pyrimidin-2-yl-thio)hexanoate) Fig.  was deveearly AD , 38.Fig.Unless otherwise stated, chemicals were obtained from Sigma-Aldrich . MH84 phenethoxy)pyrimidin-2-yl-thio)hexanoate) was synthesized according to literature procedures . Analyti1H-NMR 2SO): δ = 0.78–0.83 , 1.22–1.37 , 1.74–1.92 , 3.06–3.11 , 4.27–4.30 , 4.48–4.53 , 5.87 , 7.49–7.52 , 7.63–7.66 , 12.82 . 13C-NMR 2SO): δ = 13.64 (Bu-CH3), 21.67 (Bu-CH2), 28.92 (Bu-CH2), 30.91 (Bu-CH2), 34.16 , 47.46 (S-CH), 66.54 , 85.97 (Pyr-C5), 124.99 , 125.14–127.74 , 129.69 , 143.06 , 168.95 (Pyr-C2), 169.97 , 172.23 (COOH). MS (ESI): m/e = 601.4 (M–1). Anal. (C28H28F6N2O4S (602.17)) C, H, N, S: ber, C 55.81, H 4.68, N 4.65, S 5.32; gef, C 56.10, H 4.86, N 4.40, S 5.40; Abw, C 0.29, H 0.18, N 0.25, S 0.08.SL mice (Thy-1 AβPPSL (control) and Thy-1 AβPPSL (MH84)) [SL (MH84) mice received 12 mg/kg b.w. MH84 by oral gavage once a day for 21 days. MH84 was diluted in polyethylene glycol 400 (PEG400). Control groups received an equal amount of PEG400. Health was assessed daily and PEG400 had no adverse effects. The mice were sacrificed by decapitation after cervical dislocation and the brains were quickly dissected on ice after removal of the cerebellum, the brain stem, and the olfactory bulb. All experiments were carried out by individuals with appropriate training and experience according to the requirements of the Federation of European Laboratory Animal Science Associations and the European Communities Council Directive (Directive 2010/63/EU). Experiments were approved by the regional authority .Twenty-two C57BL/6 mice at the age of 3 months bearing the human Swedish (S: KM595/596NL) and London (L: V717I) mutations in the 751 amino acid form of human amyloid-beta precursor protein (AβPP) under control of a murine Thy-1 promoter were randomly divided into two groups of 11 Thy-1 AβPP (MH84)) . A numbe® pistil in 2 ml mitochondrial respiration medium (MiR05) containing a protease inhibitor cocktail . The resulting homogenate was centrifuged to remove the nuclei and other cell debris . The low-speed centrifugation step was repeated once (for 3 min) with the supernatant followed by a high-speed centrifugation step  to collect a pellet enriched in mitochondria. This pellet was resuspended in 1 ml ice-cold MiR05 + PI and centrifuged . The supernatant was centrifuged once again at high speed  to obtain the mitochondrial fraction. The resulting pellet was then dissolved in 250 μl MiR05 + PI. The obtained solution of isolated mitochondria was then used for high-resolution respirometry (80 μl). The remaining solution was immediately frozen in liquid nitrogen for citrate synthase (CS) activity (120 μl) and protein determinations (50 μl) and stored at –80 °C.Brain mitochondria were isolated from one hemisphere as reported previously . BrieflyOXPHOS) pyruvate (5 mM), malate (2 mM), and adenosine 5′-diphosphate (ADP) (2 mM) followed by addition of succinate . Mitochondrial integrity was examined via addition of cytochrome c (10 μM). Subsequently, oligomycin (2 μg/ml) was added to reveal leak respiration (leak omy). The maximum capacity of the electron transfer system (ETS) was achieved by titration of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). Complex II respiration in the noncoupled state (CIIETS) was measured after adding rotenone (0.5 μM) into the chambers. Residual oxygen consumption (ROX) was determined after inhibition of complex III by addition of antimycin A (2.5 μM) and was subtracted from all respiratory parameters. Cyclooxygenase activity (CIVETS) was measured after by applying 0.5 mM tetramethylphenylenediamine (TMPD) as an artificial substrate of CIV and 2 mM ascorbate to keep TMPD in the reduced state. The autoxidation rate was determined after the addition of sodium azide (≥100 mM). For data analysis, DatLab 5.1.1.7 software (Oroboros) was used. Data of mitochondrial respiration were normalized to CS activity.Mitochondrial respiration was analyzed as reported previously . Briefly2. A 1-ml aliquot of the remaining cell suspension was collected and immediately frozen at –80 °C for protein determination.Dissociated brain cells (DBC) were isolated from one hemisphere as reported previously , 40. BriThe potential of the inner mitochondrial membrane (MMP) was determined in DBC as described previously using the cell-permeable fluorescent dye rhodamine 123 (R123) with a VICTOR™ X3 2020 Multilabel Counter  .® Plus bioluminescence assay according to the manufacturer’s instructions  with a VICTOR™ X3 Multilabel Counter (Perkin Elmer), as described previously [ATP levels were determined in DBC using the ViaLighteviously , 40.® Analytics, Muellheim, Germany). CS activity was assessed at 412 nm using a GENESYS 5 spectrophotometer  and normalized to protein content. Measurements were performed in duplicate.CS activity was determined in isolated mitochondria as described previously . A frozeAβPPwt) and HEK293AβPPsw cells transfected with DNA constructs harboring human mutant AβPP  gene were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 400 μg/ml Geniticin (G418). Both cell lines were maintained in a humified incubator, gassed with 5% CO2 at 37 °C as described previously [AβPPwt cell line was a kind gift received from Prof. Christian Haass, Munich, Germany.Human embryonic kidney (HEK) 293 cells containing human wild-type AβPP (HEK293eviously . The HEKAβPPwt cells and HEK293AβPPsw cells were seeded into a 24-well plate. On the next day cell medium was incubated for 24 h with 0.1 μM MH84. For solvent control DMSO (0.05%) was used. Afterward the cells were washed with 500 μl Hank’s Balanced Salt Solution buffer (HBSS)  and spiked with 1 μM MTG  and incubated for 60 min. After adding fresh HBSS buffer to the cells, fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 516 nm in a VICTOR™ X3 Multilabel Counter (Perkin Elmer). When comparing HEK293AβPPwt cells and HEK293AβPPsw cells, fluorescence values were normalized to protein content.MitoTracker Green (MTG) measurements were performed as described previously . Briefly1–40 and Aβ1–42 in supernatants was detected by specific solid phase sandwich enzyme-linked immunosorbent assays  following the instructions of the supplier. Brain material was homogenized in 10 times the amount of phosphate-buffered saline containing a protease inhibitor cocktail. After homogenization, samples were centrifuged  and the supernatants were transferred to a fresh reaction vessel and stored at –80 °C prior to analysis. Total protein content was determined using a Pierce™ BCA Protein Assay Kit .The content of soluble Aβ® Total OXPHOS Rodent WB Antibody Cocktail . As primary antibody for synaptic markers, Anti-BDNF , Anti-GAP43 , and Anti-synaptophysin  were used. To detect protein expression of PGC-1α in the brain, Anti-PGC-1α antibody  was used. Anti-tubulin antibody  and Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody  were used to verify equal protein loading.Western blot analysis was carried out in brain tissue homogenates as reported previously . BrieflyqRT-PCR measurements were carried out as described previously . Briefly® Green Supermix, and forward and reverse primers at the following concentrations: 50 nM (PGC-1α), 200 nM (BACE1), or 400 nM . After a 3-min activation of Taq polymerase, amplification was allowed to proceed for 46 cycles. Each cycle consisted of denaturation at 95 °C for 10 s, annealing at 58 °C for 45 s, and extension at 72 °C for 29 s. BACE1 mRNA expression was performed in a two-step PCR amplification. After a 2-min activation of Taq polymerase, amplification was allowed to proceed for 40 cycles. Each cycle consisted of denaturation at 95 °C for 5 s and annealing and extension at 60 °C for 30 s. The one-cycle melting curve consisted of heating from 65 to 95 °C by 0.5 °C steps, with 5-s hold per step. Results were normalized to PGK1 and B2M as reference genes which are stably expressed in mouse brain [–ΔΔCq method with target-specific amplification efficiency.Prior optimization was conducted for each set of primers, which consisted of determining optimal primer concentration and template concentration and verifying the efficiency of the amplification. To confirm the specificity of the amplification, the PCR product was subjected to melting curve analysis and agarose gel electrophoresis. PCR amplification was performed in duplicate in a total reaction volume of 10 μl. The reaction mix consisted of 2 μl template, 5 μl iTaq™ Universal SYBRse brain . Resultst test and for multiple comparisons a one-way ANOVA with Tukey’s multiple comparison post test or a two-way ANOVA followed by Bonferroni post test was performed. p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.All data are presented as means ± SEM. For statistical analysis GraphPad Prism 5.03 software  was used. For the examination of statistical significant differences between two groups a two-sided, unpaired Student’s SL mice  were increased in brains of Thy-1 AβPPice Fig. , b. Elevice Fig. –d. Approely Fig. , f. Leveice Fig. , e. ExprSL mice  . Western1/F0-ATPase (CV)) that produces ATP. Respiration of CIV of the mitochondrial respiration chain was significantly decreased in mitochondria isolated from brains of Thy-1 AβPPSL mice ; complex II, succinate dehydrogenase (CII); complex III, cytochrome-c reductase (CIII); complex IV, cytochrome-c oxidase (CIV)) build up a proton gradient at the inner mitochondrial membrane. The resulting membrane potential (MMP) finally represents the driving force for complex V of the mitochondrial respiration chain . Decreaearly AD  and in aearly AD . PGC-1α early AD , 56. Thuearly AD  and mitoearly AD . Accordiearly AD . In our n et al.  detectedlication  and othelication –61.SL mice.Mice were treated for 21 days with 12 mg/kg body weight (b.w.) MH84 using oral gavage. Pharmacokinetic properties of MH84 were investigated previously and we reported a maximum brain concentration of 320.64 ng/g . MH84 isSL mice. These findings are in line with data for a cellular model of AD that we have been reported recently [SL mice. Enhanced MitoTracker Green (MTG) fluorescence after incubation of HEK-AβPPsw cells with MH84 further supports this hypothesis. MTG accumulates in mitochondria independently of the mitochondrial membrane potential and thus represents another suitable marker for the determination of mitochondrial content [SL. However, in our study MH84 did not reverse enhanced BACE1 mRNA levels in brains of Thy1-AβPPSL mice. On the other site, PGC-1α overexpression was reported to exacerbate Aβ and tau deposition in another transgenic mouse model of AD [The present study also shows that MH84 reversed mitochondrial dysfunction and PGC-1α levels, decreased β-secretase processing of AβPP, and reduced levels of soluble Aβ40 in brains of transgenic Thy-1 AβPPrecently . PGC-1α recently , 56. Thu content . Katsour content , 64 showel of AD .SL mice, which was reversed after treatment with MH84. Thus, effects of MH84 might at least partly be related to enhanced BDNF expression although synaptic marker proteins were not changed.BDNF represents one of the most widely distributed and extensively studied neurotrophins in the mammalian brain. Its functions include developmental processes, regulation of synaptogenesis, and neuroprotection . DecreasMH84 represents a dual γ-secretase/PPARγ modulator that improves mitochondrial dysfunction by enhancing mitochondrial dysfunction via a PGC-1α-dependent mechanism. Thus, MH84 seems to be a new promising therapeutic agent with approved in-vivo activity for the treatment of AD that could help to modify the disease progression in early stages.Additional file 1: Figure S1.Representative NMR spectra of MH84 used for the current study. For analytical details please refer to . , Thy-1 AβPPSL (control), and treatment group Thy-1 AβPPSL (MH84)). Data represent means ± SEM. N = 11 ; one-way ANOVA with Tukey’s multiple comparison post test ). (TIFF 562 kb)Brain levels of the β-site of APP cleaving enzyme (BACE-1) mRNA. Expression levels of mRNA were normalized to PGK1 and B2M mRNA expression. Animals belonged to three different study groups , Thy-1 AβPPSL (control), and MH-84-treated Thy-1 AβPPSL (MH84) mice. Tubulin  and GAPDH (B) were used as loading controls. Data represent means ± SEM. N = 11 ; one-way ANOVA with Tukey’s multiple comparison post test ; +p < 0.05 against Thy-1 AβPPSL (control)). (TIFF 12802 kb)Western blot analysis of A brain-derived neurotrophic factor (BDNF), B Growth associated protein 43 (GAP43), and C synaptophysin in brain homogenate isolated from wild-type mice


The coupling was achieved by a Suzuki–Miyaura reaction utilizing the PTABS ligand and palladium(II) acetate.Deoxyuridine substituted by dibenzofuranyl at the carbon atom in base position C 21H18N2O6, has a bent rather than a linear conformation supported by three intramolecular C—H⋯O hydrogen bonds. The packing in the crystal lattice is largely determined by interactions between hydrogen atoms with oxygen atom lone pairs with one molecule interacting with neigbouring molecules via O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds. The title compound crystallizes in the chiral orthorhombic space group P212121. Its absolute structure could not be determined crystallographically and was assumed with reference to that of the reactant 5-iodo-2′-deoxyuridine.The molecule of the title compound, C The bond lengths of the pyrimidine moiety observed for the title compound are very close to the average values found for related structures (see Table S1 in the Supporting information). As is typical for this class of compounds, the bond usually assigned to be a double bond within the six-membered ring (here C6=C7) is the shortest for the pyrimidine ring at 1.353 (6) Å and the bond between the second carbonyl carbon atom and the substituted carbon (here C7—C8) is the longest at 1.447 (6) Å. All four other ring atom-to-ring atom distances (N—C and C—C bonds) are shorter than 1.393 Å, indicating significant π-electron delocalization throughout the pyrimidine base. All this, however, is in accordance with the majority of previously reported structures.The title compound crystallizes in the orthorhombic space group )° Fig. 1. All bon6-H function (here C6) and the ring oxygen atom of the sugar (here O3) and/or the –CH3–OH group (here O1) is useful for evaluation in this context. The C—H⋯O hydrogen-to-oxygen distances for the interaction with the alcohol range from 2.29 to 5.98 Å  between the C6–H moiety points to some extent towards the methanoyl oxygen atom of the sugar, forming a weak intramolecular hydrogen bond and resulting in comparable molecular bends. The dihedral angles between the two aromatic systems do vary and range from 11.9° for a ferrocene substituent . In the c- and (by bifurcation) a-axis directions, both classical and non-classical hydrogen bonds are present . These interactions lead to the formation of slabs lying parallel to the ac plane.In the crystal, molecules are linked by N—H⋯O, O—H⋯O and C—H⋯O hydrogen bonds Fig. 2. The molet al., 20162 and PTBS (phospha-triaza-adamantyl propane sulfonate) in water.The title compound was synthesized according to our recently reported method (Bhilare Synthesis of 5-(dibenzofuran-4-yl)-2′-deoxyuridine: To a solution of palladium acetate  and PTABS ligand  in degassed water (1.0 ml) at ambient temperature under N2 were added 5-iodo-2′-deoxyuridine (0.5 mmol) and the solution stirred for 5 min at 353 K. After that, the reaction mixture was allowed to cool to room temperature and then 4-(dibenzofuranyl)boronic acid (0.75 mmol) was added along with triethylamine  and degassed water (2.0 ml). The resulting solution was then stirred at 353 K for 3 h. The reaction progress was monitored by TLC. After the completion of reaction, the solvent was removed in vacuo and the resultant residue obtained was purified using column chromatography in CH2Cl2:MeOH solvent system (96:4) to afford the desired product as a white solid .M) λabs = 286 nm λfl = 392,427. 1H NMR  δ 11.62 , 8.41 , 8.12 , 8.06 , 7.67 , 7.49 , 7.38 , 6.28 , 5.29 , 4.87 , 4.27 , 3.81 , 3.54 , 2.29–2.14 . 13C NMR  δ 161.7, 155.3, 152.9, 150.0, 140.1, 128.3, 127.6, 123.8, 123.6, 123.2, 122.8, 121.1, 120.3, 117.8, 111.7, 108.8, 87.6, 84.5, 70.5, 61.4, 39.9. ESI–MS (m/z) = 395 (M+ + H+). Analysis calculated for C21H18N2O6: C, 63.96; H, 4.60; N, 7.10. Found: C, 63.85; H, 4.64; N, 6.98.UV–visible absorption and fluorescence emission in methanol (10 µSHELXL instruction: SADI 0.05 O1 H1O O2 H2O N2 H2N), as otherwise the N—H distance became rather short and the O—H distances rather long. The respective orientations, i.e. the directions the hydrogen atoms are pointing to , were refined without any restraints or constraints. The C-bound H atoms were included in calculated positions and treated as riding: C—H = 0.95–1.00 Å with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017013111/ds2247sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017013111/ds2247Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017013111/ds2247sup3.pdfBond lengths in uracil bases with substitution at the C5 position. DOI: 1574284CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Here we show that the Vδ2+ compartment comprises both innate-like and adaptive subsets. Vγ9+ Vδ2+ T cells display semi-invariant TCR repertoires, featuring public Vγ9 TCR sequences equivalent in cord and adult blood. By contrast, we also identify a separate, Vγ9− Vδ2+ T-cell subset that typically has a CD27hiCCR7+CD28+IL-7Rα+ naive-like phenotype and a diverse TCR repertoire, however in response to viral infection, undergoes clonal expansion and differentiation to a CD27loCD45RA+CX3CR1+granzymeA/B+ effector phenotype. Consistent with a function in solid tissue immunosurveillance, we detect human intrahepatic Vγ9− Vδ2+ T cells featuring dominant clonal expansions and an effector phenotype. These findings redefine human γδ T-cell subsets by delineating the Vδ2+ T-cell compartment into innate-like (Vγ9+) and adaptive (Vγ9−) subsets, which have distinct functions in microbial immunosurveillance.Vδ2 + γδ T cells are thought to be an innate-like T-cell population. Here the authors show the Vδ2+ compartment contains both innate-like Vγ9+ and an adaptive Vγ9- subset that undergoes clonal expansion during viral infection and can infiltrate liver tissue.Human Vδ2 They provide anti-microbial2 and anti-tumour immunity3, but whether they occupy an innate-like or adaptive immunological niche has remained unclear. Notably, αβ T cells incorporate a group of unconventional T cells, including mucosal-associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cells that recognise antigens in the context of single MHC-like proteins (MR1 and CD1d), and display a semi-invariant T-cell receptor (TCR) repertoire, suggestive of an innate-like biology whereby TCR sensitivity is retained but the γδ TCR may arguably function as a surrogate pattern recognition receptor4. Notably, studies in mice have suggested that innate-like γδ T-cell development in the thymus can occur via distinct pathways involving agonistic signals5. In addition, recently, Wencker et al.6 have suggested that following TCR triggering during development, mouse innate-like T cells may transition to a state of TCR hyporesponsiveness in which they preferentially respond to TCR-extrinsic stimuli such as cytokine exposure.γδ T cells have coevolved alongside B cells and αβ T cells in the vertebrate immune system for almost 450 million years+ and Vδ2− subsets7. Vδ2− γδ T cells have been directly implicated in anti-viral and anti-tumour immunity8 and utilise germline-encoded antigen receptors also present on innate-like lymphocytes, including NKG2D and NKp3010. However, recent evidence has suggested that they may adopt a TCR-dependent adaptive immunobiology, based on highly clonotypically focused expansions alongside differentiation from a naive to effector phenotype11 and perturbations in clonal expansion upon cytomegalovirus (CMV) infection in post-stem cell transplant patients12. Conversely, Vδ2+ T cells are arguably the prototypic unconventional T cell, typically co-expressing Vγ9 TCR chains and representing the major γδ subset in adult peripheral blood13. Vγ9+ Vδ2+ T cells respond to prenyl pyrophosphate metabolites  produced either by the host mevalonate pathway  or microbial non-mevalonate pathway ((E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, HMB-PP)14, which are sensed in the context of butyrophilin 3A1 (BTN3A1)17. They mount important anti-microbial immune responses, including against Mycobacterium tuberculosis13 and Plasmodium falciparum18, and drive αβ T-cell responses20.Human γδ T cells are often delineated into Vδ2+ Vδ2+ T cells observed in adult peripheral blood, which is proposed to result from exposure to P-Ag-producing microbes encountered after birth22 and thought to involve clonotypic expansion24, arguably suggests an adaptive immunobiology. However, Vγ9+ Vδ2+ T cells are highly enriched in foetal peripheral blood and display restricted complementarity-determining region (CDR) 3 γ9 usage early in gestation25, more consistent with an innate-like pre-natal repertoire of Vγ9+ Vδ2+ T cells.Previously, the extrathymic expansion of Vγ9+ γδ T-cell immunobiology we carried out a dedicated analysis of their clonotypic diversity and cellular phenotype. Our findings suggest Vδ2+ T cells can be delineated into two discrete subsets: Vγ9+ Vδ2+ T cells adopt a predominantly innate-like biology originating in neonatal development and allowing a degree of clonotypic plasticity, whereas Vγ9− Vδ2+ T cells adopt a distinct adaptive immunobiology, including focused clonal expansions and differentiation evident both in peripheral blood and solid tissues, and generated in response to acute viral infection. These findings revise our understanding of human γδ T cells, and prompt further investigation of the adaptive immunity provided by the Vγ9− Vδ2+ subset.To better understand human Vδ2+ γδ T cells were present at relatively low levels, but were increased as a proportion of peripheral T cells by adulthood26  and TCRδ (between 1.8 and 39%) repertoires  of the Vδ2+ T-cell repertoire  than expanded Vδ1 clonotypes . Unlike the Vδ1 compartment, similarly prominent clonotypes were also present in cord blood Vδ2+ TCRγ (between 18 and 34%) repertoires  and clonally expanded adult Vδ1 repertoires , although this proportion was increased in cord blood CDR3δ repertoires , representing a mean 38.9% of the total CDR3γ9 repertoire Fig. . This hial Table , and are+ T cells from each donor to establish the relationship with TCR clonotype. Adult Vγ9+ Vδ2+ T cells from healthy donors  regularly display a major CD27+ CD45RA− phenotype  CD8+ αβ T cells) and a minor CD27− CD45RA− phenotype (similar to Effector Memory (EM) CD8+ αβ T cells)  Fig. , consistD27 Fig.  and distD27 Fig. .+ Vδ2+ TCR diversity to cord blood samples, two adult donors (6 and 24) appeared to exhibit a degree of clonotypic focusing, based on their low D75 values for both TCRγ (mean 2.9) and TCRδ (mean 5.0) combined with their higher cumulative frequency of the 10 most prevalent clonotypes (mean 60.5% of TCRγ and mean 50.3% of TCRδ)  in the majority of donors, there is scope within the Vγ9+ Vδ2+ TCR repertoire for a degree of specific clonal amplification, alongside concomitant phenotypic changes.Although overall our adult cohort displayed similar Vγ9Rδ) Fig. . ConsistRδ) Fig. . Both doRδ) Fig. . Also, dRδ) Fig. . Furtherype Fig. . These d+ T cells were dominated by Vγ9 TCR sequences, but we noted the presence of non-Vγ9  sequences in adult TCRγ repertoires (mean 4.46%)  , while this population was undetectable using another TCR Vδ2 antibody clone, B6 (Biolegend), which is likely to be specific for the Vγ9+ Vδ2+ TCR pairing  Fig. . Althouglls Fig.  and quanlls Fig. . Importa2%) Fig. , but on 2%) Fig. . Notablying Fig. .Fig. 3Vγ+ T cells are commonly responsive to host and microbial P-Ags, we assessed the proliferative capacity of Vγ9− Vδ2+ T cells towards microbial P-Ag (HMB-PP). While CD8+, both Vδ2+ subsets and Vδ1+ T cells all proliferated in response to anti-CD3/CD28 stimulation, only Vγ9+ Vδ2+ T cells responded to HMB-PP  but together highly similar to CD27hi Vδ1+ T cells . Importantly, and in contrast to the dominant CD27hi phenotype displayed by Vγ9− Vδ2+ T cells in all other healthy donors, “donor X’s” Vγ9− Vδ2+ T-cell population had downregulated CD27 and retained CD45RA expression (CD27lo/neg phenotype), now accounting for 91.4% of the Vγ9− Vδ2+ T-cell population , composed of a Vδ2 CDR3 (CACGSWWGTYTDKLIF) paired with a Vγ8–JγP1 chain , CX3CR1, Grz A and had downregulated CCR7, in contrast to naive CD8 T cells, but closely matching CD27lo/neg Vδ1+ T cells  had a substantially increased Vγ9lls Fig. , far higion Fig. . We nextain Fig. , and nowire Fig. . As thislls Fig. , which a+ T cells are predominantly associated with a role in tissue immunity36. To address whether the Vγ9− Vδ2+ T-cell population was also represented in solid tissues, we analysed human liver samples for the presence of intrahepatic Vγ9− Vδ2+ T cells  infection, previously shown to drive the numeric37 and clonal12 expansion of Vδ1+ T cells. We examined a cohort of five CMV-seronegative (CMV−) patients receiving CMV-seropositive (CMV+) kidney transplants who then went on to either develop post-operative acute CMV infection , or who remained CMV− (patient 279 and 282)38. We analysed peripheral blood samples for Vγ9− Vδ2+ T cells before and after post-transplant CMV infection. Prior to CMV infection, at 0–4 weeks post-transplant, Vγ9− Vδ2+ T cells represented low frequency populations in all donors  and were stable for over 5 years (patient 231)  Fig. . These d+ T cells, highlighting divergent Vγ9+ and Vγ9− subsets. Despite the ~ten-fold increase in Vγ9+ Vδ2+ T-cell numbers in the first year of life, our data indicate that the extent of clonotypic focusing within the Vδ2-associated Vγ9+ repertoire is broadly similar between neonatal cord blood and adulthood. This finding contrasts with the Vδ1+ TCR repertoire, which typically displays pronounced clonotypic focusing in adults relative to a highly unfocused neonatal repertoire11. In addition, our analyses reveal an underappreciated degree of public amino acid sequences in the Vγ9 repertoire. This degree of Vγ publicity, which relates to both recombination of simple Vγ9 sequences (either germline encoded or with limited N nucleotide addition) and convergent recombination, underlines the semi-invariant nature of the Vγ9+ Vδ2+ TCR repertoire. Importantly, using single-cell approaches public Vγ clonotypes were shown frequently to pair with diverse Vδ2 chains within single individuals, and, therefore, do not usually represent expanded clonotypes. The innate-like biology of the semi-invariant Vγ9+ Vδ2+ T-cell subset contrasts markedly with the Vδ1+ subset, which displays clear hallmarks of adaptive immunity such as clonal amplification and differentiation from an initially unfocused, private TCR repertoire.This study sheds light on the biology of Vδ2+ Vδ2+ T cells from foetal development into adulthood. First, as previously suggested by Dimova et al.25, we propose gestational selection of the Vγ9+ Vδ2+ subset, potentially via exposure to endogenous P-Ag  most likely mediated by BTN3A140, allowing pre-programming of a P-Ag reactive Vγ9+ Vδ2+ TCR repertoire. In keeping with this, we observed that Vγ9 chains are heavily enriched for the JγP segment, which is not generally present in Vγ9 chains present in Vδ1+ TCR repertoires11; similarly the Jδ1+ CDR3δ2 repertoire in cord blood is enriched for a hydrophobic amino acid at position 5 required for P-Ag recognition29, but not in Vδ2 sequences in Vγ9− Vδ2+ T cells (see below). Second, in the context of the reported lack of thymic output of Vδ2+ T cells after birth41, our observation that adult and cord blood repertoires contain overlapping canonical P-Ag-sensing Vγ9 sequences strongly indicates there is polyclonal post-natal expansion of this pre-selected repertoire, most likely in response to microbial P-Ag exposure11. However, our observation that cord blood Vδ2 sequences generally utilised Jδ3 segments , whereas adult Vδ2 sequences preferentially used Jδ1 segments, suggests that post-natal microbial exposure may impose some constraints on the TCR repertoire that differ from those required in gestation. Of note, Vδ2–Jδ1 sequences typically contain a hydrophobic amino acid previously highlighted as important for Vγ9+ Vδ2+ T-cell reactivity to microbially-derived P-Ag29 whereas this is less conserved in Vδ2–Jδ3. This might imply Vγ9+ Vδ2+ T cells bearing Jδ1 clonotypes preferentially respond to microbial P-Ag after birth compared to Jδ3 clonotypes, a possibility which future studies can address. Finally, although most individuals’ Vγ9+ Vδ2+ T cells were dominated by public Vγ9 sequences and associated with a common central memory phenotype, our observation that a minority of individuals exhibit amplification of unusual public clonotypes, concomitant with adoption of an effector memory phenotype, most likely reflects a third level of selection that imparts a degree of plasticity in this fundamentally innate-like paradigm. This observation extends previous work by Ryan et al.33 indicating that further functional and transcriptional changes are linked to an effector memory phenotype in Vγ9+ Vδ2+ T cells. Therefore, the semi-invariant Vγ9+ Vδ2+ repertoire may provide scope for selective TCR-mediated responses to microbial P-Ag. Importantly, our model shares a number of features with MAIT cells, a semi-invariant population that is also subject to both gestational selection (through interactions with MR1) and post-natal expansion in response to microbial infection/colonisation43, and that displays clonotype-specific responses to metabolite ligands44 and microbial species45.Collectively, our repertoire data suggest a model involving a multi-layered selection of Vγ9− Vδ2+ T cells that is highly distinct from the Vγ9+ Vδ2+ P-Ag-reactive subset, and present in all adult peripheral blood and cord blood, where they commonly exist as a CD27hi, TCR diverse subset phenotypically equivalent to naive-like Vδ1+ T cells. We note this distinction based on Vγ9 chain usage cannot be applied to the γδ T-cell compartment as a whole, as the Vδ1+ T-cell subset, which appears to exhibit an adaptive immunobiology, contains a sizeable portion of Vγ9+ T cells11. Moreover, consistent with a distinct biology for Vγ9 chains in these two contexts, the Vγ9 CDR3 sequences associated with Vδ1 chains did not utilise JγP sequences and were highly distinct from those associated with Vδ2 chains11. Consistent with expression of TCRγ and TCRδ sequences divergent from the Vγ9+ Vδ2+ subset, the Vγ9− Vδ2+ population was not P-Ag responsive. Thus, Vγ9− Vδ2+ T cells represent a universal γδ subset that is clonotypically, phenotypically and functionally divergent from Vγ9+ Vδ2+ T cells. Moreover, as evidenced from a single unusual peripheral blood donor and several liver samples, Vγ9− Vδ2+ T cells appeared capable of undergoing an adaptive-like programme of highly focused clonotypic expansion and concomitant phenotypic differentiation that closely matches that of Vδ1+ T cells but is not observed in Vγ9+ Vδ2+ T cells. This considerably extends the findings of Ravens et al.12, who detected unusual Vγ9− Vδ2+ clonotypes in the peripheral blood of one healthy donor and one HSCT recipient.Our analyses also highlight a population of Vγ9− Vδ2+ subset after infection, linked to the selection of dominant clonotypes, which occurred concomitantly with differentiation from a naive CD27hi to a CD27lo/neg phenotype, and acquisition of CX3CR1 and cytotoxic granzymes, similar to CMV-specific cytotoxic CD8+ T cells47. Interestingly, the single healthy donor who exhibited a clonally expanded Vγ9− Vδ2+ T-cell subset (“donor X”) was CMV+ and unusually, displayed elevated IgG titres, raising the possibility of recent CMV reactivation. Our findings therefore suggest a role for the Vγ9− Vδ2+ T-cell subset in unconventional immunosurveillance against viral infection, and provide a clear indication that this subset assumes an adaptive immunobiology, highly similar to that suggested for Vδ1+ T cells12. These data suggest clonal selection from a CCR7+, potentially lymphoid tissue homing, naive-like Vγ9−Vδ2+ T-cell population that expresses a pool of TCRs, enabling amplification of responses to microbial challenges, via differentiation of cells bearing biologically relevant clonotypes to an effector phenotype, resulting in long-lived, functional γδ T-cell memory. The liver provided an attractive human model in which to examine Vγ9− Vδ2+ T cells in solid tissues, as it functions as an important site for clearance of both commensals and pathogens from portal circulation, and has a high proportion of unconventional T cells, such as MAIT cells48. Our finding that clonally expanded effector Vγ9− Vδ2+ T cells can be recruited to the liver suggests this subset may complement the innate-like recognition provided by such hepatic semi-invariant T cells with an unconventional adaptive memory response. Unfortunately, we were unable to obtain CMV serostatus for the patients from whom explanted liver samples were derived, and therefore unable to conclude if the presence of such hepatic Vγ9− Vδ2+ T cells solely reflected CMV seropositivity or alternatively a wider set of immune challenges. Of relevance, a monoclonally expanded myocytotoxic T-cell population bearing a Vγ3+ Vδ2+ TCR detected in a patient with polymyositis highlights the potential of the Vγ9− Vδ2+ T-cell subset to trigger autoimmunity50. Further studies are required to define additional microbial and non-microbial challenges that stimulate this unconventional T-cell subset.This adaptive biology was confirmed by our analysis of patients following acute CMV infection, which highlighted a striking expansion of the Vγ9− Vδ2+ T cells were TCR responsive but unresponsive to IL-12/IL-18 is another feature that distinguishes them from Vγ9+ Vδ2+ T cells, and one that again aligns them closely with the Vδ1+ T-cell subset, which has also been highlighted to display adaptive features12 Further studies on such adaptive subsets (Vδ1+ and Vγ9− Vδ2+ T cells), ideally ultimately in the context of physiological TCR ligands, are required to explore the full range of functional differences with innate-like subsets. Of note, Vγ9+ Vδ2+ T cells have previously been demonstrated to exert pleiotropic effector functions in response to cognate P-Ag ligands and different cytokine stimulation51. Moreover, recent studies in mice highlight that γδ TCR triggering of innate-like lymphocytes induced a state of TCR hyporesponsiveness in which T-cell activation could be induced by TCR-extrinsic stimuli6 Unfortunately, a systematic comparison of the TCR responsiveness of naive vs. effector Vγ9− Vδ2+ T cells within one tissue was not possible, due to the strong dominance of naive Vγ9− Vδ2+ T cells in peripheral blood and effector Vγ9− Vδ2+ T cells in liver. However, importantly within the Vδ1+ T-cell compartment, CD27lo/neg effectors display a far quicker response to TCR stimulation than CD27hi naive cells11. This observation emphasises the strong distinction between innate-like and adaptive subsets within the γδ T-cell compartment.In addition to clonal expansion in response to CMV, the finding that peripheral blood Vγ9+ T cells therefore comprise two separate subsets, which reflect distinct paradigms in human γδ T-cell biology and also have distinct roles in antimicrobial immunity. Vγ9+ Vδ2+ T cells appear to represent an innate-like subset, most likely originating via gestational selection, which is ‘pre-armed’ from birth with a semi-invariant TCR repertoire that includes a high level of public Vγ TCR chains, permitting polyclonal TCR-mediated responses to microbial P-Ag, as well as to innate cytokines independently of the TCR. However, the Vγ9+ Vδ2+ TCR repertoire diversity may provide the potential for clonotype-specific responses potentially targeted at specific microbial antigenic challenges. In contrast, Vγ9− Vδ2+ T cells, which are P-Ag-unreactive, represent a previously ill-defined unconventional T-cell compartment that exhibits many of the key hallmarks of an adaptive immunobiology, including clonal expansion and differentiation to effector lymphocytes from an initially naive-like and TCR diverse T-cell pool, including in response to acute viral infection. In doing so, Vγ9− Vδ2+ T cells most likely contribute to both peripheral blood and tissue immunosurveillance.Human Vδ2-4 copies/ml, the MMF dose was halved and Valcyte was given. Only patient 1014 experienced CMV-related symptoms: CMV gastritis. All but one patient were treated with a basic immunosuppressive regimen comprising of CD25 mAb (basiliximab) induction treatment, prednisolone, cyclosporine A and MMF. Patient 261, however, was treated with one dose of prednisolone upon transplantation with a kidney from an identical twin, whereafter all immunosuppression was ceased. Liver infiltrating T cells were isolated from explanted diseased human liver tissues from patients undergoing liver transplantation for inflammatory liver diseases, including primary biliary cholangits, primary sclerosing cholangitis, alcoholic liver disease, and autoimmune hepatitis  or normal liver samples from donor liver tissue surplus to clinical requirements . Umbilical cord blood units were obtained from the Anthony Nolan Cell Therapy Centre Nottingham (ANCTC) under generic tissue bank ethics held by ANCTC and extended to the researchers under a material transfer agreement (MTA).Peripheral blood samples were obtained from healthy donors who had provided written informed consent for sample collection and subsequent analysis; project approval for this aspect of the study was granted by the NRES Committee West Midlands ethical board (REC reference 14/WM/1254). Samples from patients undergoing renal transplantation were obtained at the Academic Medical Centre, Amsterdam; the medical ethics committee of the Academic Medical Center, Amsterdam, approved this arm of the study and all subjects provided written informed consent in accordance with the Declaration of Helsinki. Longitudinal heparinized blood samples were collected from CMV-seronegative patients who developed primary CMV infection after receiving a renal transplant from a CMV-seropositive donor. As controls, longitudinal PBMC from age matched CMV-seronegative patients who remained CMV-seronegative during the first 5 years following renal transplantation were used. All patients were EBV-seropositive. The course of CMV infection was followed by longitudinal 2-weekly PCR for CMV viral load, and seroconversion was confirmed by detection of CMV-specific IgM and IgG. The patients did not receive CMV prophylaxis, but once the CMV load reached 10PBMC were isolated from heparinised venous blood by lymphoprep© (Stem Cell Technologies) density gradient centrifugation as per the manufacturers instructions. For proliferation of T cells, PBMC were labelled with 0.3 µM carboxyfluorescein succinimidyl ester (CFSE) (eBioscience) and cultured with 10 nM HMB-PP (Sigma) or CD3/CD28 T activator beads (Invitrogen) for 7 days in RPMI-1640 medium (Invitrogen) supplemented with 2 mM L-glutamine, 1% sodium pyruvate, 50 μg/ml penicillin/streptomycin (Invitrogen) and 10% fetal calf serum (Sigma).+ populations, PBMC were labelled with anti-CD3 , TCR αβ , TCR Vδ2 , CD27 , CD45RA ; both Biolegend, and populations were sorted on an ARIA III Fusion (BD) . For phenotypic analysis, freshly isolated or frozen PBMC, or cultured cells were labelled with Zombie Aqua viability dye (Biolegend), and then subsequently stained , CD16 , CD69 , TCR Vδ2 , TCR αβ ; all Biolegend. TCR Vγ9 ; Beckman Coulter. TCR Vδ1  and TCR Vδ2 ; Miltenyi, or αGalCer loaded CD1d dextramers . For intracellular staining, after surface antibody staining cells were fixed in IC Fixation buffer (eBioscience) and stained in Permeabilisation Buffer (eBioscience) with antibodies directed against Granzyme A  and Granzyme B ; Biolegend). Cells were acquired on an LSR Fortessa X20 (BD) and data analysed with FlowJo V10.2 (TreeStar).For total and single-cell sorting of Vδ2+: 25,000 cells; cord blood Vδ2+: 2,400 - 8,800 cells) using an RNAmicro plus kit (Qiagen) according to the manufacturer’s instructions. For high throughput deep sequencing of γδ TCRs, we used amplicon rescued multiplex (ARM)-PCR and a MiSeq (Illumina) next-generation sequencer (NGS)52 to analyse all sorted Vδ2+ T-cell populations. As detailed in patent WO2009137255A2, a modified version of a protocol devised by Han et al.53 was used, involving initial first-round RT-PCR using high concentrations of gene-specific primers, followed by use of universal primers for the exponential phase of amplification, allowing deep, quantitative and non-biased amplification of TCR γ and TCR δ sequences. All cDNA synthesis, amplification, NGS library preparation and sequencing were performed by iRepertoire, Inc. . We analysed positively sorted αβ TCR- Vδ2+ γδ T cells from 7 healthy donors and 4 umbilical cord blood units .RNA was purified from sorted cells  containing 0.1% Triton X-100, and incubated according to manufacturer’s instructions. TCRγ and TCRδ cDNAs were amplified by two rounds of nested PCR using GoTaq mastermix (Promega) and primers for Vδ2, TCTGGGCAGGAGTCATGT  and GAAAGGAGAAGCGATCGGTAAC ; for Cδ GCAGGATCAAACTCTGTTATCTTC  and TCCTTCACCAGACAAGCGAC ; for Vγ1–8 CTGGTACCTACACCAGGAGGGGAAGG  and TGTGTTGGAATCAGGAVTCAG ; for Vγ9 AGAGAGACCTGGTGAAGTCATACA  and GGTGGATAGGATACCTGAAACG  and for Cγ CTGACGATACATCTGTGTTCTTTG  and AATCGTGTTGCTCTTCTTTTCTT . PCR products were separated on 1.2% agarose gels, and products of successful reactions were incubated with ExoSAP-IT PCR cleanup enzyme (Affymetrix) before sequencing with BigDye Terminator v3.1 (Applied Biosystems) following manufacturer’s instructions and running on an ABI 3730 capillary sequencer .PBMC were labelled as above and Vδ254. Tree maps show each unique CDR3 as a coloured rectangle, the size of each rectangle corresponds to each CDR3s abundance within the repertoire and the positioning is determined by the V region usage. To determine the 10 most prevalent shared CDR3γ9 sequences, the first 10 most dominant sequences by frequency were filtered from each donors CDR3γ9 protein lists. These sequences were then ordered by frequency for sequences that were shared between >2 donors (to create a hierarchy of 10 common sequences) and uncommon highly amplified sequences were included in the final analysis. For more detailed analysis of the TCR repertoire, datasets were processed using the MiXCR software package55 to further correct for PCR and sequencing errors. Diversity metrics, clonotype overlap and gene usage were plotted in R, by VDJTools56.V, D and J gene usage and CDR3 sequences were identified and assigned and tree maps generated using iRweb tools 58. Neonatal Vδ2 sequence logos were generated on the Seq2Logo server59 in Shannon format without the use of pseudo counts, and give a visual representation of amino acids enriched at different positions in the observed CDR3δ2 sequences. The different amino acids are coloured according to physicochemical properties (acidic (DE), red; basic (RKH), blue; hydrophobic (ACFILMPVW), black; and neutral (NSGTY), green). For TCRδ2 sequences, the ten most abundant clonotypes of 14–16 amino acids using either Jδ3 or Jδ1 from each donor were aligned using Clustal Omega52 with default parameters, before logo generation. Narrower bars in the sequence logo correspond to gaps in the sequences.The CDR3 sequence was defined as the amino acids between the second Cysteine of the V region and the conserved Phenylalanine of the J region, according to IMGT. CDR3 sequences shown in tables include the conserved Cysteine and Phenylalanine, but only the amino acids between these residues are counted for CDR3 length analysis and for analysis of residues at position 5. N and P nucleotides were identified using the IMGT Junction Analysis toolt-tests for normally distributed data and Mann–Whitney for non-parametric data. Differences between groups were analysed using one-way ANOVA with Dunnett’s or Tukey’s post tests for normally distributed data or with Kruskal–Wallis with Tukey’s post tests for non-parametric data and RM two-way ANOVA with Tukey’s post tests was used when comparing groups with independent variables. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Correlation was assessed for non-parametric data by Spearman correlation. Tabulated data were analysed in Graphpad PRISM 7 .Each data set was assessed for normality using Shapiro–Wilk normality test. Differences between columns were analysed by two-tailed Student’s SRP113556 and SRP096009, for Vδ2+ and Vδ1+ TCR repertoires respectively.All data are available from the authors upon request. The sequence data that support the findings of this study have been deposited in the NIH NCBI sequence read archive (SRA) database with the primary accession code Supplementary Information


UGT79B31 encodes flavonol 3-O-glycoside: 2″-O-glucosyltransferase, an enzyme responsible for the terminal modification of pollen-specific flavonols inPetunia hybrida.P. hybrida) and maize (Zea mays). As a first step toward elucidating the role of flavonoids in pollen, we have identified a glycosyltransferase that is responsible for the terminal modification of petunia pollen-specific flavonoids. An in silico search of the petunia transcriptome database revealed four candidate UDP-glycosyltransferase (UGT) genes. UGT79B31 was selected for further analyses based on a correlation between the accumulation pattern of flavonol glycosides in various tissues and organs and the expression profiles of the candidate genes. Arabidopsis ugt79b6 mutants that lacked kaempferol/quercetin 3-O-glucosyl(1 → 2)glucosides, were complemented by transformation with UGT79B31 cDNA under the control of Arabidopsis UGT79B6 promoter, showing that UGT79B31 functions as a flavonol 3-O-glucoside: 2″-O-glucosyltransferase in planta. Recombinant UGT79B31 protein can convert kaempferol 3-O-galactoside/glucoside to kaempferol 3-O-glucosyl(1 → 2)galactoside/glucoside. UGT79B31 prefers flavonol 3-O-galactosides to the 3-O-glucosides and rarely accepted the 3-O-diglycosides as sugar acceptors. UDP-glucose was the preferred sugar donor for UGT79B31. These results indicated that UGT79B31 encodes a flavonoid 3-O-glycoside: 2″-O-glucosyltransferase. Transient expression of UGT79B31 fused to green fluorescent protein (GFP) in Nicotiana benthamiana showed that UGT79B31 protein was localized in the cytosol.Flavonoids are known to be involved in pollen fertility in petunia (The online version of this article (10.1007/s00425-017-2822-5) contains supplementary material, which is available to authorized users. Flavonoids are major plant secondary metabolites with over 9000 compounds distributed widely throughout the plant kingdom Markham . The bioP. hybrida) are unable to germinate, resulting in male sterility  mutants deficient in flavonoids are also male sterile galactosides that are identical to those accumulating in the wild type glucoside are prominent flavonoids in maize pollen glucosides (3-O-sophorosides) are the dominant flavonol glycosides in pollen from Arabidopsis thaliana , the enzyme responsible for the terminal modification of pollen flavonoids, respectively glucosides by UGT79B6, a flavonol 3-O-glucoside: 2″-O-glucosyltransferase. UGT78D2 is expressed throughout the plant but UGT79B6 expression is specific to the tapetum and microspores of developing anthers  galactosides by an unknown flavonol 3-O-galactoside: 2″-O-glucosyltransferase (F3GT)  were used. Petunia seeds were sown on one-half-strength MS-agar medium containing 2% (w/v) sucrose and placed in a 25 °C growth chamber with a light intensity of 70 µmol of photons m2 s−1 and a 16 h light/8 h dark photoperiod. After 8 weeks, the seedlings were transferred to sterile vermiculite and acclimated. After acclimation, plants were transferred to soil and grown for 3 months in a greenhouse.Seeds of Arabidopsis thaliana accession Columbia-0  was used as the wild type. The Arabidopsis TILLING line CS95581 (ugt79b6-3) was previously described  at the Sol Genomics Network (https://solgenomics.net/). Sequences with the following scores were used for further analyses: Categories/Database, Transcriptome projects/P. hybrida var. Mitchell transcriptome Villarino 2014 Contigs, > 200; SGN Unigenes current version/P. hybrida Unigenes > 80; SGN ESTs/P. hybrida SGN mRNA sequences > 80.A BLAST search with the tblastn program was conducted using https://solgenomics.net/) was conducted as follows: Categories, Petunia sps. Genome (current version); Database, Petunia axillaris v1.6.2 proteins/Petunia inflata v1.0.1 proteins; Program, blastp. Sequences showing over 40% identity with the query were used for the analyses.For a comprehensive search for homologues of UGT79B31, UGT92A1 and SGN210759, a BLAST search in the Sol Genomics Network : scan duration, 1.0 s; inter-scan delay, 0.1 s. MS/MS data were acquired in the ramp mode in the following analytical conditions: (1) MS: mass range, m/z 50–1500; scan duration, 1.0 s; inter-scan delay, 0.014 s; data acquisition, centroid mode; and (2) MS/MS: mass range, m/z 50–1500; scan duration, 0.1 s; inter-scan delay, 0.014 s; data acquisition, centroid mode; collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra of the top 10 ions  in an MS scan were automatically obtained. If the ion intensity was less than 10,000, MS/MS data acquisition was not performed and moved to the next top 10 ions.Fresh samples were extracted with 5 μl of 80% MeOH containing 2.5 µM lidocaine and 10-camphor sulfonic acid per mg fresh weight using a mixer mill with zirconium beads for 7 min at 18 Hz and 4 °C. After centrifugation for 10 min, the supernatant was filtered using an HLB μElution plate (Waters). The extracts (1 μl) were analyzed using LC-QTOF-MS . Analytical conditions were as follows: LC column, Acquity bridged ethyl hybrid (BEH) C18 ; solvent system, solvent A [water including 0.1% (v/v) formic acid] and solvent B [acetonitrile including 0.1% (v/v) formic acid]; gradient program, 90% A/10% B at 0 min, 90% A/10% B at 0.1 min, 80% A/20% B at 25 min, 0% A/100% B at 25.1 min, 0% A/100% B at 27.5 min, 90% A/10% B at 27.6 min and 90% A/10% B at 30.0 min; flow rate, 0.3 ml/min; column temperature, 40 °C; photodiode array, 200–500 nm; flavonoid detection, 320 nm; MS detection: capillary voltage, + 3.0 keV, cone voltage, 25.0 V, source temperature, 120 °C, desolvation temperature, 450 °C, cone gas flow, 50 l/h; desolvation gas flow, 800 l/h; collision energy, 6 V; mass range, P. hybrida V26 anthers at developmental stages 2 and 3  with thermal cycling conditions as follows: PCR mixture was incubated at 98 °C for 10 s, followed by 30 cycles of PCR , and finally incubated at 72 °C for 3 min. The resultant product (ca. 700 bp) was cloned and sequenced.Complementary DNA from poly(A) + RNA isolated from d 3 Fig. a was synUGT79B31 is described in the next section. Real-time PCR was performed on three biological samples.RNA extraction and cDNA synthesis were performed as described previously , petunia pollen cDNA and primers Phcomp17948_(−1)f and Phcomp17948_1368r (Table S1); the amplification product was cloned into the pCR2.1-TOPO vector. The sequence of the resultant plasmid, pKYS479, was confirmed to exclude PCR errors. To construct the protein expression vector, full-length UGT79B31 was further PCR amplified using primers, UGT79B31/pColdProS2f and UGT79B31/pColdProS2r (Table S1) and pKYS479 as a template; the amplification product was cloned into pColdProS2 using an In-Fusion Advantage PCR Cloning Kit (Clontech). After verifying the sequence of the resultant plasmid, pKYS491, the plasmid was transformed into E. coli strain BL21star™ (DE3). Production and purification of the recombinant protein were performed as described previously  according to the manufacturer’s instructions. The ProS2 tag was removed using HRV3C protease (Novagen) according to the manufacturer’s instructions. After exchanging the buffer for 50 mM Hepes–KOH, pH 7.5, proteins were concentrated using an Amicon Ultra filter .Full-length The standard enzyme assay reaction mixture was described previously  promoter region was amplified with the primers At5g54010promoter-683 and At5g54010promoter-R (Table S1) and cloned into the pENTR/D-TOPO vector to construct the plasmid pKYS449 . Plasmids pGWB1 and pKYS492 were used for LR reactions to construct the binary vector pKYS498 using Gateway LR Clonase™ II Enzyme Mix (Invitrogen). Plasmid pKYS498 (pGWB1/683 bp fragments of the UGT79B6 promoter fused to UGT79B31 CDS) was used to transform Agrobacterium and Arabidopsis ugt79b6 mutants as described previously . Three days after infiltration, leaves were analyzed using a Zeiss LSM700 inverted confocal laser scanning microscope with a 40× dry objective (Zeiss). A diode laser with 488 nm excitation and appropriate filters (505–600 nm) were used to detect GFP fluorescence. ZEN 2011 software (Zeiss) was used for data analysis.UGT79B31 was amplified with primers, GFP-79B31f/GFP-79B31r and 79B31-GFPf/79B31-GFPr (Table S1) for fusion to N- and C-terminal GFP, respectively; the amplification products were cloned into the pGWB5 or pGWB6 vectors .Tobacco leaves (~ 10 mg) sampled 3 days after infiltration were ground in 150 µl sample buffer  containing 5% β-mercaptoethanol. Samples were denatured at 98 °C for 2 min and proteins were separated by 10% polyacrylamide gel  electrophoresis. Proteins were transferred to PVDF membranes  by semidry electroblotting. Blots were probed with anti-GFP primary antibody  and alkaline phosphatase conjugated anti-mouse secondary antibody  and visualized using WesternBluehttps://solgenomics.net/) using UGT79B6 from A. thaliana as a query. To date, the identified flavonoid glycosyltransferases that catalyze glycosylation of the sugar moiety attached to flavonoid aglycones (GGTs) belong to the UGT79 and UGT94 families. The UGT79, UGT91, UGT92 and UGT94 families belong to the same orthologous group that contains genes derived from a common ancestor (Yonekura-Sakakibara and Hanada P. hybrida (Phcomp61074_c0_seq1 and Phcomp17948_c0_seq2) (Table S1). The amplified product (668 bp) corresponded to Phcomp17948_c0_seq2. Phcomp61074_c0_seq1, Phcomp17948_c0_seq2 and Phcomp27832_c0_seq1 were designated as UGT79D1, UGT79B31 and UGT91S1, respectively, by the UGT nomenclature committee . Thus, we obtained four candidate genes  in total.To identify the UGT(s) catalyzing the terminal glucosylation of pollen-specific flavonols, we conducted an in silico search of the Sol Genomics Network database (O-glucosyl(1 → 2)galactosides accumulated exclusively in the pollen of P. hybrida V26 (Vogt and Taylor O-glucosyl(1 → 2)glucoside derivatives were identified in petals and leaves of the Petunia cultivar ‘Mitchell’ galactoside/glucoside, the flavonoid distribution in anthers, petals and young leaves of P. hybrida V26 was determined. Kaempferol/quercetin 3-O-glucosyl(1 → 2)galactoside (F1/F2) were confirmed to accumulate in the anthers of P. hybrida V26 glucoside (F3) and the compounds with an m/z value corresponding to quercetin 3-O-glucosyl(1 → 2)glucoside (F4) were detected in petals of P. hybrida V26, and a negligible amount of kaempferol/quercetin 3-O-glucosyl(1 → 2)galactosides (F1/F2), kaempferol 3-O-glucosyl(1 → 2)glucoside (F3) and the compound with the same m/z value corresponding to quercetin 3-O-glucosyl(1 → 2)glucoside (F4) were also detected in leaves glycosides. Therefore, we selected UGT79B31 for further analysis.Expression profiles of the four UGT genes in various organs, tissues and developmental stages of petunia were analyzed by quantitative reverse transcription PCR Fig. . The UGTE. coli and was partially purified. After cleavage of the His/ProS2-tag, the UGT79B31 protein was used in enzyme assays. The recombinant UGT79B31 protein converted kaempferol 3-O-galactoside to kaempferol 3-O-glucosyl-(1 → 2)-galactoside as confirmed by comparable retention times and MS spectra as pollen kaempferol 3-O-glucosyl-(1 → 2)galactoside -glucoside by UGT79B31 was confirmed glucosides were significantly lower than those for the 3-O-glucosides, suggesting that UGT79B31 had a low affinity for the 3-O-diglycosides as sugar acceptors -glucosides. UGT79B31 can utilize flavonol 3-O-glucosides in addition to the 3-O-galactosides as substrates in vitro, suggesting that UGT79B31 may complement the ugt79b6 mutation. The ugt79b6 mutants were transformed with the full-length UGT79B31 cDNA coding region under the control of the Arabidopsis UGT79B6 promoters (pKYS498). The flower flavonol profiles of transgenic ugt79b6-3 lines transformed with pKYS498 were completely restored to that of wild type  and slightly lower identity among other known flavonoid GGTs, IpA3G2″GlcT (52.5%), UGT79B30 , UGT79B6 , UGT79B1 , CsF7G6″RhaT (43.9%), UGT79A6 , GmF3G6″GlcT  and PhA3G6″RhaT (38.9%). The relatively lower sequence identity with flavonol 3-O-galactoside/glucoside: 2″-O-glucosyltransferase from soybean  suggests that flavonoid GGTs may gain their substrate specificity in a plant lineage-specific manner.Generally flavonoid UGTs form a distinctive cluster based on the position of sugar acceptors they glycosylate. UGT79B31 belongs to the same cluster as other known flavonoid GGTs (OG8 cluster) and to a subcluster containing flavonoid glycoside: 2″-ily Fig. . UGT79B3P. hybrida is derived from a white-flowered P. axillaris and a purple-flowered species of the P. integrifolia clade  using UGT79B31, UGT92A1 and SGN210759 as queries. Most UGTs from P. axillaris and P. inflata are in a one-to-one correspondence, indicating that gene duplication of UGTs in OG8 did not occur frequently after the divergence of P. axillaris and P. inflata because the two species separated relatively recently. UGT79B31 had the highest sequence identities with Peaxi162Scf00248g00003.1 (99.56%) and Peinf101Scf00058g07001.1 (98.02%) from P. axillaris and P. inflata, respectively, suggesting that these two genes may be UGT79B31 orthologs.The commercial N. benthamiana galactoside in petunia is first determined by a glycosylation step catalyzed by F3GalT, not UGT79B31. In contrast, UGT78D2 which catalyzes the first 3-O-glucosylation in Arabidopsis is distributed nearly throughout the plant and UGT79B6 expression is specific to pollens Supplementary material 2 (DOCX 25 kb)Below is the link to the electronic supplementary material.


The EF-hand type calcium-binding protein S100A12 exerts numerous intra- and extracellular functions of (patho)physiological relevance. Therefore, receptors of S100A12 are of high interest for research and clinical applications. Beside the extensively studied receptor for advanced glycation endproducts (RAGE), G-protein coupled receptors and more recently, scavenger receptors are suggested to be putative S100A12 receptors. Own findings and further information from the literature predestined CD36, a class B scavenger receptor, as promising candidate. To substantiate or prove against this hypothesis, this study aimed at investigation of interaction of S100A12 and CD36 on molecular and cellular level by the use of surface plasmon resonance (SPR), radio- and fluorescence-tracer-based cell binding, and cell activation experiments. S100A12 revealed binding affinity to CD36 in the low nanomolar range, essentially, at the CD36 thrombospondin-1 binding site. Additionally, S100A12-mediated translocation of CD36 to the membrane and elevation of both CD36 and peroxisome proliferator-activated receptor γ (PPARγ) expression was observed, which suggest a potential regulatory function of S100A12–CD36 interaction.The online version of this article (doi:10.1007/s00726-016-2349-2) contains supplementary material, which is available to authorized users. Positive clones were checked for success of cloning and transfection using colony-PCR and sequencing analyses (Agowa GmbH). Transfection of plasmid-DNA into CHO-cells was performed using Lipofectamine™ 2000. Transfection was performed in serum free medium with 1 µg of plasmid-DNA and for 6–8 h at 37 °C under normal cell culture conditions . Subsequently, cells were incubated in serum containing medium with selected antibiotics (300 µg/mL G418 and 250 µg/mL Zeocin). The success of transfection of CHO-RAGE cells was checked using fluorescence microscopy, Western blot analyses and flow cytometry.Chinese hamster ovary (CHO)-K1 and CHO-CD36 cells were purchased from ATCC. CHO-RAGE cells were generated as described elsewhere  and anti-goat AlexaFluor594® (for anti-RAGE antibody) were used. Counterstaining was conducted using the cell DNA marker Hoechst 33258 (5 µg/mL). Images were acquired using the confocal laser-scanning microscope IX83 (Olympus).Synthesis of CD36 and RAGE was detected by immunocytochemical staining. Therefore, cells were fixed with 4 % (w/v) paraformaldehyde and 2.5 % (w/v) sucrose in phosphate buffered saline (PBS). After permeabilization with 0.3 % (v/v) Triton-X-100 in PBS, unspecific binding sites were blocked with a blocking solution containing 5 % (w/v) bovine serum albumin (BSA) and 0.5 % (v/v) Tween 20 in PBS. For antibody staining, the monoclonal anti-CD36 antibody [FA6-152]  and the polyclonal anti-RAGE antibody  as well as secondary antibodies, anti-mouse AlexaFluor4882, 1.3 mM CaCl2; pH 7.5).Western blot analyses were performed as published elsewhere  of 1.0 was reached, protein expression was induced by adding 0.5 mmol/L isopropyl-β-d-1-thiogalactopyranoside (IPTG) for 4 h at 25 °C. The cell pellet was lysed using 20 % (v/v) Triton X-100, 4000 U/mL lysozyme, 25 U/mL benzonase and ultrasound. RS100A12 protein was purified using glutathione-sepharose. Glutathione S-transferase (GST) free proteins were eluted using PreScission™ protease . Further purification was performed using size exclusion chromatography. The purified protein was analyzed by mass spectrometry and Western blot . After snap freezing in liquid nitrogen, the purified protein was stored at -65 °C until usage for further experiments. Furthermore, commercially available native human S100A12 protein (nS100A12) was obtained from Life Technologies with the reference number 11143-HNAE-50. Compared to the purchased native nS100A12 the recombinant synthesized protein rS100A12 possesses five additional N-terminal amino acids (GPLGS)  was performed as published elsewhere  was performed as published by us earlier for the investigation of the interaction of S100A12 and RAGE -rS100A12 was purified using a HiTrap desalting column  with the ÄKTAprime® plus  chromatography system. The activity of the product was determined using the ISOMED 2000 calibrator (Nuklear-Medizintechnik Dresden GmbH).Radiolabeling of rS100A12 with N-hydroxysuccinimide-fluorescein  was used by following the manufacturer’s instruction. Briefly, 1 mg of rS100A12,  in borate buffer (pH 8.0) was reacted with 15 times molar excess of NHS-fluorescein  for 1 h at room temperature. Purification of the fluorescein-rS100A12 was performed in Slide-A-Lyzer™ dialysis cassettes (Thermo Fisher) with an exclusion size of M < 7000 g/mol. Depending on the following study PBS or calcium binding buffer (20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 150 mM NaCl, 1.2 mM MgCl2, 1.3 mM CaCl2; pH 7.5) were used.To obtain a second, labeled S100A12 protein species, as independent marker for cell binding experiments and SPR studies (as described below), fluorescein-labeled rS100A12 was synthesized. Therefore, 18F]FB-rS100A12 binding cells were incubated with [18F]FB-rS100A12 in PBS++ for 90 min at 37 °C. For blocking, a 15-fold excess of rS100A12 related to [18F]FB-rS100A12 or the monoclonal anti-CD36 antibody   Protein Assay (Thermo Scientific) following the manufacturer’s instruction. The amount of cellular fluorescein-rS100A12 binding was determined similar to the [18F]FB-rS100A12 experiments. Cells were grown in 24-well plates to 80 % confluence. Cells were incubated with 200 µL of incubation solution for 90 min at 37 °C. For antibody blocking experiments, the monoclonal anti-CD36 antibody  was added to the cells for 30 min at 37 °C in a concentration of 10 µg/mL before starting the cell binding experiment. Afterwards, cells were lysed with sodium hydroxide  and SDS [1 % (w/v)]. Fluorescence signal quantification was checked with assays using cell lysates using the Synergy™ 4 Multi-Mode Microplate Reader (BioTek) with λEx = 485 nm, λEm = 525 nm as filter setting. Obtained relative fluorescence units were put in relation to the amount of protein in the cell lysate. Protein determination was performed using the Pierce® BCA Protein Assay (Thermo Scientific) following the manufacturer’s instruction.To quantify the amount of FB-rS100A12 to the CHO cell lines at 37 °C is shown. Compared to CHO-K1 cells, cell association of [18F]FB-rS100A12 was significantly higher by 40 % in CHO-RAGE cells and by 20 % in CHO-CD36 cells. On the other hand, using the CD36-specific antibody (FA6-152) binding of [18F]FB-S100A12 to CHO-CD36 cells could be substantially blocked. Therefore, a specific binding of [18F]FB-S100A12 to CD36 could be confirmed. The fact that [18F]FB-rS100A12 was blocked by unlabeled rS100A12 indicates a similar binding behavior of both the unlabeled and the labeled species.In Fig. As independent marker for cell binding fluorescein labeled S100A12 was used. Therefore, experiments were performed with 10 µM of fluorescein-S100A12. Compared to CHO-K1 cells, binding was significantly higher by 50 % in CHO-RAGE and by 30 % in CHO-CD36 cells  and commercially available nS100A12  to CD36 did not differ significantly and were determined to be close to 1 nM. In comparison, fluorescein-labeled rS100A12 showed a weaker binding affinity (KD = 4.79 ± 0.38 nM) to CD36.SPR analyses were performed to characterize binding of S100A12 to CD36 at the protein–protein interaction level. Human recombinant CD36  was chosen as ligand and immobilized on the sensor surface by EDC/NHS amine coupling chemistry. A blank immobilized sensor surface served as reference cell to account for non-specific binding effects. Recombinant rS100A12, commercially available human, untagged recombinant nS100A12, and fluorescein-labeled rS100A12 were used as ligands. Experiments were performed as single cycle kinetics and analytes were used in a concentration range of 0.62–50.0 nM  could not be observed in CHO-K1, CHO-CD36, and CHO-RAGE cells by immunoblotting . However, incubation of CD36-positive CHO cells with rS100A12 lead to the recruitment of CD36, which normally is diffusely distributed in vesicles, to the cell surface Fig. a, b. On 18F] fluorobenzoylated and fluorescein-labeled rS100A12 showed that S100A12 specifically binds to cells expressing CD36. Similarly, although in a higher amount, [18F]FB-rS100A12 binds to cells expressing RAGE, a well-characterized receptor of S100A12. Blocking with a CD36-specific monoclonal antibody [FA6-152] that specifically blocks the collagen and thrombospondin binding sites of CD36 Below is the link to the electronic supplementary material.


The benzoquinoxaline ring system is almost planar (r.m.s. deviation = 0.076 Å). A pseudo-twofold rotation axis runs through the midpoints of the C2—C3 and C9—C10 bonds. The two phenyl rings are twisted relative to the benzoquinoxaline ring system, making dihedral angles of 53.91 (4) and 36.86 (6)°. In the crystal, C—H⋯π (arene) interactions link the molecules, but no π–π interactions between aromatic rings are observed.The title compound, C The packing in the crystals is described as having a herringbone motif benzoquinoxaline ligand. Finally, in the square-planar PtII complex benzoquinoxaline. In the distorted octahedral Co1H NMR spectra were recorded with a 300 MHz Varian Mercury model in CDCl3. 4,4-Dimethylbenzil (3 mmol), 2,3-diaminonaphthalene (4.4 mmol), and iodine (0.37 mmol) were dissolved slowly in acetonitrile (10 ml), and stirred for 10 minutes at room temperature. The reaction mixture was poured into water, extracted with ether and dried over anhydrous MgSO4. After volatiles had been removed under reduced pressure, the product was purified by silica gel chromatography using an eluent of hexane/ethyl acetate (20:1). Pale-yellow single crystals of the title compound were obtained from dichloromethane/hexane (1:1) solution within a few days by slow evaporation of the solvent at 298 K, yield: 48%. 1H NMR : 8.72 , 8.12 , 7.56 , 7.49 , 7.18 , 2.40 .Chemicals were obtained commercially in reagent grade and used as received. Solvents were dried using standard procedures as described in the literature. d(C—H) = 0.93–0.96 Å, and with Uiso(H) = 1.2Ueq(C) for aromatic-H and 1.5Ueq(C) for methyl-H atoms, respectively.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018004413/vm2210sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018004413/vm2210Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018004413/vm2210Isup3.cmlSupporting information file. DOI: 1543571CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Induced CD4 + and CD8+ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8− DCs were critical for priming CD4+ T cell responses.Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4 DCs are critical for the induction of protective immune responses to pathogens as well as for the maintenance of tolerance to self- and innocuous foreign antigens . Therefo+ or CD4+ T cell responses upon specific loading with antigen in vivo  are important for the recognition and clearance of IgG opsonized microorganisms by phagocytes, but they also enhance antigen uptake and presentation by DCs and macrophages . To date in vivo . Further in vivo .+ and CD8+ T cell responses under steady-state conditions dependent on either CD11c+CD8− or CD11c+CD8+ DCs, which in both cases ultimately resulted in T cell number reduction. Under inflammatory conditions, however, antigens taken up via the activating FcγRIV demonstrated superior CD4+ and CD8+ T cell responses in vivo. Interestingly, these T cell responses were independent of FcγR ITAM signaling, demonstrated using NOTAM mice mutated in the ITAM of the Fcε receptor γ-chain  is an activating receptor, whereas the other (FcγRIIB) is the inhibitory FcγR. This allows for the study of whether Fc receptor signaling has an impact on the ensuing T cell response. In addition, we used an antibody specific for FcγRIIB/FcγRIII to target one inhibitory receptor and one activating Fc receptor in parallel. For the genetic fusion of the model antigen ovalbumin (Ova) with FcγR-specific antibodies, we cloned the variable regions of the αFcγRIIB, αFcγRIIB/III, and αFcγRIV antibodies. We further changed the constant part of the heavy chains (HCs) to a modified mouse IgG1 fragment incapable of being bound by FcγRs to prevent an antibody Fc-dependent interaction with other Fc receptors and genetically fused these antibodies with Ova, as we have done previously for the DC-specific targeting antibodies αDEC205 and αDCIR2 .+CD8+ DCs were more efficient in the uptake of FcγR-targeting antibodies than CD11c+CD8− DCs. In addition, we found no differences in the uptake of FcγR antibodies when compared with each other. αDEC205- and αDCIR2-targeting antibodies were internalized as expected from previous results  into various splenic cell populations, we labeled the antibodies with a specific oligo harboring an Atto647N dye as previously reported . After i results . The tartibodies .+ or CD4+ T cells. Therefore, we transferred congenic antigen-specific CFSE-labeled CD4+ or CD8+ T cells into C57BL/6 mice followed by injection of different amounts of endotoxin-free αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova antibodies 16 h later  resulted in either no or only slight differences in the induced T cell responses in NOTAM mice compared with wild-type C57BL/6 mice. This suggests that ITAM signaling has no major influence on the T cell responses induced by antigen-targeting antibodies specific for activating FcγRs ; αCD40/pIC) as described before (9 heat-killed Listeria monocytogenes (HKLM). Interestingly, 3 d after priming, the αDEC205-Ova targeting was superior in the induction of CD8+ T cell responses in the absence of adjuvant, whereas all CD8+ T cell responses were comparable when αCD40/pIC was injected additionally . We also studied the DC maturation state and found no up-regulation of co-stimulatory molecules by DT injection . Next, congenic antigen-specific CD8+ , we were interested in FcγRIV as a potential co-stimulator for other targeting antibodies. We therefore injected all Ova-containing antibodies with 20-fold excess of an FcγRIV targeting antibody without Ova (αFcγRIV w/o Ova). However, this did not result in increased T cell responses upon targeting of FcγRIIB, FcγRIIB/III, and DCIR2. As expected, the additional injection of αFcγRIV w/o Ova blocked the response by FcγRIV-Ova targeting. In contrast, we observed a slight increase of the CD8+, but not the CD4+ T cell response induced by αDEC205-Ova targeting when we coinjected the αFcγRIV w/o Ova antibody, which will be subject to further investigations  into the very same animals. 16 h later, the efficacy of antigen-specific target cell lysis was measured by flow cytometry. We found efficient target cell lysis after immunization with αDEC205-Ova, αFcγRIIB/III-Ova, and αFcγRIV-Ova, whereas targeting by αFcγRIIB-Ova was slightly less and αDCIR2-Ova least efficient , FcγRIV (activating), and FcγRIIB/III (inhibitory and activating).Targeting antigens to DCs in vivo provides an efficient means to induce CD4esponses . Besidesesponses . However+ DCs and some select DC subsets or receptors  but also the environment (steady state vs. proinflammatory) determines whether antigen uptake via FcγRs on DCs will result in short-term or long-lasting T cell responses. On a cellular level, the DC subset and not the type of receptor will govern whether a CD4−/−, CD11c-Cre, LysM-Cre, CD45.2+ OT-I, and CD45.2+ OT-II mice were obtained on C57BL/6 background from The Jackson Laboratory. OT-I and OT-II mice were crossed with CD45.1+ B6.SJL-PtprcaPep3b/BoyJ mice to a CD45.1 background. FcγRI−/− (R1 B6), FcγRIIB−/− (R2 B6), FcγRIII−/− (R3 B6), FcγRIV−/− (R4 B6), and FcγRIVfl/fl mice were generated in the laboratory of Jeffrey Ravetch and are on the C57BL/6 background (The Rockefeller University). FcγRIVfl/fl animals were crossed to LysM-Cre and CD11c-Cre mice to generate FcγRIVfl/fl × LysM-Cre and FcγRIVfl/fl × CD11c-Cre mice, respectively. NOTAM mice were generated in the laboratory of Jeanette Leusen on C57BL/6 background and backcrossed at least 15 times to C57BL/6 FcRγ−/− mice . αFcγRIIB and αFcγRIV hybridoma cells were cultured in ISF-1 medium (Biochrom) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin.HEK293T cells were cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM 3) or MACS buffer .Single-cell suspensions from mouse spleen tissue were obtained by digestion with (FACS) or without  Collagenase D (Serva) and DNase I (Roche) as described before . Erythro2 α–mouse-IgG1 human adsorbed: PE), αArHamIgG (goat α–ArmenianHamster IgG: PE). Isotype controls used were ArHam-PE (clone eBio299Arm: PE), msIgG1 , msIgG2a , ratIgG2a , ratIgG2b . Other stains used were DAPI, PI, Strepavidin-PerCP-Cy5.5, Strepavidin-PE-Cy7, Strepavidin-V500, and FcγRIIB/III , FcγRIIB , and FcγRIV  for blocking.If not otherwise stated, antibodies were purchased from eBioscience, BD, BioLegend, SouthernBiotech, or Invitrogen or produced in-house. Used antibodies were CD3ε (clone eBio500A2: eFluor450), CD4 (clone RM4-5: APC-eFluor780), CD8α , CD11b , CD11c , CD16 , CD16/32 , CD19 (clone eBio1D3: eFluor450), CD25 (clone 3C7: APC), CD32 , CD40 (clone 3/23: PE), CD44 , CD45R , CD45.1 (clone A20: eFluor450), CD49b , CD62L (MEL-14: PE), CD64 , CD69 (clone H1.2F3: PE-Cy7), CD80 , CD90.1 (clone HIS51: eFluor450), CD205 , CD317 , CD335 , DCIR2 , FcγRIV , INFγ , Ly6C , Ly6G , MHC-II , Siglec-H (clone eBio440C: FITC), TCR-Vα2 . Secondary antibodies used were αmsIgG1 (goat F(ab′)3) on collagenase D/DNase I–treated single-cell suspensions. Where applicable, unwanted binding of FACS antibodies to Fc receptors was prevented by blocking of FcγRIV with the 9E9 blocking antibody in addition to blocking FcγRIIB and FcγRIII by 2.4G2 (Bio X Cell) for 15 min at 4°C. In the first staining step, cells were incubated with all directly labeled and biotinylated antibodies for 15 min at 4°C. After washing, biotinylated antibodies or unlabeled antibodies were visualized using conjugated Streptavidin or secondary antibodies for 15 min at 4°C, respectively. For the detection of FcγRs, first the specific FcγR antibody was stained combined with an excess of blocking antibodies for the remaining FcγRs  for 15 min at 4°C. Afterward, all other antibodies were stained as described above.All FACS steps were performed in FACS buffer , instantly cooled down on ice, and directly fixed with 1% formaldehyde for 10 min on ice. Afterward, αmsIgG1-PE was applied as secondary antibody. After intensive washing, cells were stained with cell identification antibodies as described above.Endocytosis assays were performed on directly isolated, Collagenase D (Serva)–, and DNase I (Roche)–treated single-cell suspensions from spleens. For the indirect endocytosis assay, splenocytes were stained with 2 µg/ml αFcγRIIB-Ova  or 1 µg/ml anti–FcγRIV-Ova  after blockade of FcγRIV and FcγRIII  or FcγRIIB and FcγRIII , respectively, in FACS buffer for 20 min on ice. After intensive washing, cells were resuspended in RMPI1640 supplemented with 5% FCS, 2 mM 3 oligo (Biomers.net) for 16 h. The labeled antibodies were purified using a ZEBA desalting column . To study their internalization, Collagenase D– and DNase I–treated splenic single-cell suspensions were incubated with the labeled antibodies in RMPI1640 supplemented with 5% FCS, 2 mM L glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM β-mercaptoethanol for 0, 10, 30, 60, or 120 min at 37°C. Extracellular fluorescence was quenched by adding an excess of a 5′-AGCCGAGGGTCCTGAACTGA-3′-BBQ650 oligo (Biomers.net). After washing, cells were stained with the cell identification antibodies as described before.To assess the uptake of the antibodies to directly compare the efficiency of the select receptors and the respective antibodies used for antigen targeting in this study, we applied a new labeling and quenching technique developed by Liu et al. . In brieAll FACS analyses were performed on a FACS-Canto II (BD) and LSR Fortessa (BD), and cell sorts were performed on a FACSAria II (BD) or MoFlo (Beckman Coulter) system. Data were analyzed with DIVA (BD) and FlowJo (Tree Star) Software.18-Primer (5′-TTTTTTTTTTTTTTTTTTVN-3′). After tailing of the gene-specific cDNA by TdT (Thermo Fisher Scientific) with dG or dA (Roche), a PCR with a proofreading enzyme  using a second nested gene specific primer  and an abridged anchor primer (5′-CAGATCGGCCACGCGTCGACTAGTATTTTTTTTTTTTTTTTTTTTTVN-3′ or 5′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′) was performed to amplify the DNA, which was then cloned into a blunt topoisomerase vector pSC-B-amp/kan . After sequencing, cloning product specific forward and reverse primers  were designed to amplify the variable regions of the HCs and light chains (LCs) from the whole cDNA pool. Overlap PCRs were used to generate fusion constructs of the original variable regions and a mutated (N297A) mouse IgG1 constant region (Fc receptor antibodies αFcγRIIB (CT17.2), αFcγRIV (9E9), and αFcγRIIB/III (2.4G2) were cloned from the original hybridoma cells. RNA was prepared by QIAGEN RNeasy Mini kit and reverse transcribed into specific cDNA by using Superscript III (Invitrogen) and gene-specific primers  or an anchored dTt region , which it region  to generChimeric antibodies were produced by transient transfection of HEK293T cells as described before . In brieSDS-PAGE was performed as described before . In brie+ OT-I or CD45.1+ OT-II mice by positive selection of CD8+ or CD4+ T cells using paramagnetic beads (Miltenyi Biotec). Purity (≥95%) was assessed regularly by flow cytometry. After CFSE labeling (Thermo Fisher Scientific), 106 CD8+ OT-I or 2 × 106 CD4+ OT-II T cells were i.v. injected into congenic CD45.2+ recipient mice. 16 h later, targeting antibodies were i.p. injected in different amounts as indicated in the experiments. Mice were sacrificed, and splenocytes were analyzed for proliferation of the transferred transgenic T cells. In case of depletion of specific cell populations, mice were i.v. injected with PBS or 20 ng/g body weight of DT (Merck or Sigma-Aldrich) 24 h before antibody injection. For depletion of pDCs, 200 µg αPDCA-1 antibody was i.p. injected 72, 48, and 24 h before injection of the targeting antibodies.Ova-specific transgenic T cells were isolated from CD45.1+ and CD115+ cells, as well as CD19+ B cells by MACS beads (Miltenyi Biotec). CD11c+CD115+ cells were further separated by cell sorting on a FACS-Aria-II or a MoFlo system (Beckman Coulter) into lin− (CD3−CD19−NKp46−Ly6G−Siglec-H−) CD11bhighCD11clowLy6Chigh, lin−CD11bhighCD11cint Ly6Clow, lin−CD11bnegCD11chighCD8+, and lin−CD11bposCD11chighCD8− cells. Cell sort purified cells and CD19+ MACS bead–enriched B cells were irradiated (3 Gy) and co-cultured with positive MACS bead–enriched CD8+ OT-I or CD4+ OT-II T cells. 16 h (OT-I) or 40 h (OT-II) later, 3H-thymidine was added to the cultures. T cell proliferation was assessed by measurement of 3H-thymidine incorporation 24 h later.C57BL/6 mice were i.p. injected with 10 µg (or 30 µg) αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova (30 µg), αFcγRIV-Ova, or 30 µg isotype-Ova control antibodies. 12 h later, mice were sacrificed, and single-cell suspensions from spleens were positively enriched for a mixture of CD11cl-glutamine, 5% heat-inactivated FCS (Biochrom), 50 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM β-mercaptoethanol in 48-well plates. C57BL/6 mice were i.p. injected with 10 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova in the presence of 50 µg αCD40 antibody (clone 1C10) and 25 µg poly(I:C) (InvivoGen) or 109 HKLM. 14 d later, 105 freshly isolated MACS bead–enriched CD11c+ cells from naive C57BL/6 animals were loaded with an overlapping Ova peptide pool (Miltenyi Biotec) according to the manufacturers’ instructions, 2 µM peptide 75 , and 2 h later, 5 µg/ml cytokine-trapping Brefeldin A (BD) was added. A stimulation with 1 µg/ml αCD3 antibody and 2 µg/ml αCD28 antibody served as a positive control and stimulation with αCD28 antibody alone as a negative control. After 8 h, cells were washed and subjected to intracellular cytokine staining. For this purpose, cells were fixed and permeabilized according to the manufacturer’s protocol by Perm/Fix and Perm/Wash , respectively. Cells were stained with antibodies for B220, CD4, CD8, and IFNγ.The whole assay was performed in RPMI1640 medium supplemented with 2 mM + B6.SJL-PtprcaPep3b/BoyJ mice and generated 10 differentially labeled cell populations. Therefore, we first labeled the cells with two different concentrations of CFSE (3 µM or 15 µM) for 20 min, 37°C in the dark as described before . Specific lysis was calculated by using two different non-SIINFEKL-loaded populations accounting for injection variances and unspecific lysis of the target cells  in PBS with 50 µg αCD40 antibody (clone 1C10) and 25 µg poly(I:C) (InvivoGen). Eight days later, mice were challenged with a mixture of the prepared target cells by i.v. injection of 10et cells .L. monocytogenes 10403S was harvested in late log phase, centrifuged, and washed three times in PBS. The recovered bacteria were resuspended in PBS and incubated at 70°C for 20 min. Bacterial concentration before killing and absence of viable bacteria after heating were monitored by counting the CFU on brain–heart infusion agar plate. The 1.0 × 1010 HKLM/ml suspension was stored at −80°C.To prepare the HKLM, a bacterial culture of Fig. S1 shows the gating strategy of DC subpopulations and other cells in mouse spleen. Fig. S2 shows depletion of cDCs by DT injection in CD11c-DTR transgenic mice and pDC depletion by αPDCA-1 injection in C57BL/6 mice.