Halide abstraction chemistry is performed on a series of trigonal lanthanide(III) complexes to provide bent complexes that contain only two N-donor ligands. 2+ complexes with only two monodentate ligands have been isolated, examples in the most common +3 oxidation state have remained elusive due to the greater electrostatic forces of Ln3+ ions. Here, we report bent Ln3+ complexes with two bis(silyl)amide ligands; in the solid state the Yb3+ analogue exhibits a crystal field similar to its three coordinate precursor rather than that expected for an axial system. This unanticipated finding is in opposition to the predicted electronic structure for two-coordinate systems, indicating that geometries can be more important than the Ln ion identity for dictating the magnetic ground states of low coordinate complexes; this is crucial transferable information for the construction of systems with enhanced magnetic properties.Low coordinate metal complexes can exhibit superlative physicochemical properties, but this chemistry is challenging for the lanthanides (Ln) due to their tendency to maximize electrostatic contacts in predominantly ionic bonding regimes. Although a handful of Ln Ln3+ complexes with only two monodentate ligands have remained elusive to date as more Lewis acidic Ln3+ centres favour higher coordination numbers.1The isolation of low coordinate Ln complexes is often synthetically challenging, as the predominantly ionic bonding regimes in these systems favour high coordination numbers to maximize the number of electrostatic interactions between ligand donor atoms and relatively large Ln cations.1-Sm can be easily oxidized by a variety of reagents to afford heteroleptic Sm3+ halide complexes [Sm{N(SiiPr3)2}2(X)] .3+ complexes [Ln{N(SiiPr3)2}2][B(C6F5)4]  by an analogous halide abstraction from [Ln{N(SiiPr3)2}2(X)], 2][B(C6F5)4]; 3-Tm and 3-Yb are prepared by the oxidation of [Ln{N(SiiPr3)2}2]  with tBuCl and [FeCp2][PF6], respectively. We have probed the electronic structures of these exotic yet structurally simple complexes by magnetic and EPR methods, supported by ab initio calculations. This allows us to probe the effect of approximately linear, bent or planar geometries on the ligand field splitting. Simple electrostatic arguments2-Ln and 3-Ln should have opposite senses of magnetic anisotropy for a given 4fn configuration: we find that this is not always the case, and in fact can vary markedly with the degree of bending of the N–Ln–N angle.In 2018, some of us showed that 2}2][BC6F4]  in toluene gave the heteroleptic Ln3+ complexes 3-Ln in good yields (58–72%) following recrystallization from hexane (2+ bis(silyl)amide complexes have recently been applied by Anwander and co-workers.3+ analogue 3-Eu could not be accessed by analogous methods, with crystals of 1-Eu the only isolable product from numerous attempts to oxidize 1-Eu with tBuCl, [FeCp2][PF6] and Ph3CCl. This can be attributed to the preference of Eu to exhibit the +2 oxidation state over all other Ln, as illustrated by standard reduction potentials, Eθ, Ln3+ → Ln2+: –0.35 V (Eu), –1.15 V (Yb), –1.55 V (Sm), –2.3 V (Tm).3-Ln using [H(SiEt3)2][B(C6F5)4] in benzene  or toluene (Yb) yielded the bent Ln3+ complexes, 2-Ln, in moderate yields (46–70%) after recrystallization from DCM layered with hexane amide ligand. For both 2-Tm and 2-Yb only one broad peak was observed at 25.04 ppm and 11.02 ppm, respectively, which we tentatively assign to the methyl protons as these are more numerous than methine protons. No signals were observed for 2-Ln by 29Si{1H} and 13C{1H} NMR spectroscopy. Similarly, no signals were observed for the [B(C6F5)4]– anion in the 13C{1H} NMR spectra of 2-Ln; however for 2-Sm, 2-Tm and 2-Yb, the 11B{1H} NMR spectra displayed sharp peaks at –16.76, –12.35 and –14.67 ppm, respectively. The 19F{1H} NMR spectra of 2-Sm and 2-Yb each displayed three signals characteristic of the [B(C6F5)4]– anion , but only one signal was observed in the 19F{1H} NMR spectrum of 2-Tm (–128.51 ppm). No signals corresponding to 3-Ln could be seen in the 1H or 13C{1H} NMR spectra for all 3-Ln, with only diamagnetic impurities observed; no features were seen in the 19F NMR spectrum of 3-Yb. Given the paucity of information that could be extracted by NMR spectroscopy for 2-Ln and 3-Ln, we did not conduct variable temperature studies as these did not prove fruitful for 1-Ln previously;The paramagnetic Ln2-Ln and 3-Ln were determined by single crystal X-ray diffraction. Complexes 2-Tm and 3-Tm are depicted in 2-Ln are structurally analogous, though 2-Sm and 2-Yb both adopt the P21/n space group and 2-Tm crystallizes in P1[combining macron], and one molecule of DCM was present in the crystal lattice for both 2-Tm and 2-Yb, but is absent in crystals of 2-Sm. The [Ln{N(SiiPr3)2}2]+ cations in 2-Ln exhibit bent geometries defined by the two Ln–N bonds, with N–Ln–N angles of 131.02(8)° for 2-Sm, 125.49(9)° for 2-Tm, and 127.7(2)° for 2-Yb, which are in contrast to the near-linear geometries seen for 1-Ln (range 166.01(14)–175.5(2)°).2-Ln to the Ln3+ cations being more Lewis acidic than the Ln2+ centres in 1-Ln,3+ centres to form additional stabilizing electrostatic contacts with methyl and methine groups of the {N(SiiPr3)2} ligands. A permanent dipole is formed between the two formally anionic N– centres and Ln3+ ion upon bending; such dipolar stabilization mechanisms have previously been used to explain the pyramidal geometries of some f-block tris-silylamides.2-Yb (see below).The solid state structures of ge 166.01–175.5(2)ge 166.01–175.5(2)1-Ln series,3+ centres in 2-Ln exhibit more bent N–Ln–N angles, which we again ascribe to the greater charge density of smaller Ln3+ cations driving stronger electrostatic interactions with ligand C–H bonds. The {N(SiiPr3)2} ligands in 2-Ln are staggered with respect to each other, with the mean Ln–N bond lengths decreasing with Ln3+ atomic radii: 2.243(4) Å (Sm), 2.156(3) Å (Tm) and 2.148(6) Å (Yb). It may appear counterintuitive that the Ln–N bonds in 2-Ln are shorter than those in 1-Ln (2.483(6) Å, Sm; 2.373(2) Å, Tm; and 2.384(3) Å, Yb)2-Ln compared with 1-Ln, but shorter Ln–N bonds for 2-Ln are expected from an increase in Ln oxidation state. Three Si–C bonds are oriented towards the Ln3+ centre in each [Ln{N(SiiPr3)2}2]+ cation; these are assigned as Ln···Cγ–Siβ agostic-type interactions by analogy with those discussed for three-coordinate silyl-substituted Ln complexes.e.g. for 2-Tm: range Tm···C: 2.731(3)–3.051(3) Å; range Tm···H: 2.200–2.495 Å; range Tm···Si: 3.066(2)–3.178(2) Å; mean β-Si–C: 1.938(3) Å; range other Si–C: 1.889(3)–1.917(3) Å]. The [B(C6F5)4]– anions do not coordinate; the shortest Ln···F distance for 2-Yb is 4.627(4) Å, whereas for 2-Sm and 2-Tm the shortest Ln···F distances are longer at 7.957(2) Å and 7.715(2) Å, respectively. Using the IUPAC definition of coordination number as the number of metal–ligand σ-bonds,2-Ln can be considered to be formally two-coordinate as they each exhibit two Ln–N bonds; we probed the numerous additional Ln···Cγ–Siβ electrostatic interactions further through calculations as these could affect the magnetic properties of the proposed [Dy{N(SiiPr3)2}2]+ cation (see below).As with the 3-Sm has previously been reported,3-Tm and 3-Yb as all three complexes are structurally similar. Complex 3-Yb crystallizes in P1[combining macron], whilst 3-Sm and 3-Tm are in the P21/c space group. Complexes 3-Ln all crystallize with distorted trigonal planar geometries, with the Ln3+ centres positioned out of the plane defined by the two nitrogen atoms and halide (distances of Ln from N2(X) plane: 0.245(2) Å for 3-Sm, 0.3292(9) Å for 3-Tm and 0.312(2) Å for 3-Yb). As expected the Yb–F bond length of 3-Yb [1.983(2) Å] is shorter than the Ln–Cl bond lengths of 3-Sm (2.5813(7) Å) and 3-Tm (2.4832(5) Å) due to the smaller size of the fluoride anion; this also leads to differing N–Ln–N angles (3-Sm: 128.24(7)°; 3-Tm: 129.39(5)°; 3-Yb: 138.71(9)°). The mean Sm–N bond length of 3-Sm (2.306(3) Å) is significantly longer than the mean Ln–N bond lengths of 3-Tm (2.229(3) Å) and 3-Yb (2.231(4) Å), which corresponds with earlier Ln3+ ions being larger.3-Ln are longer than those in 2-Ln, as expected from increasing the formal coordination number from two to three. Finally, as with 2-Ln the coordination spheres of the Ln3+ centres of 3-Ln are completed by multiple electrostatic contacts with methine and methyl groups. These are also likely to arise from Ln···Cγ–Siβ agostic-type interactions, though in 3-Ln there are fewer, and the Tm···C/H/Si distances are generally longer due to the presence of a halide .The structure of 2-Sm, 2-Tm and 2-Yb in DCM are pale red, green and purple, respectively, and their electronic absorption spectra are dominated by strong ligand to metal charge transfer bands tailing in from the UV region (2-Sm (4f5) exhibits the most intense absorption in the visible region , whilst 2-Tm and 2-Yb exhibit weaker visible absorptions . Weak absorptions (ε < 100 mol–1 dm3 cm–1) were seen for all 2-Ln in the near-IR region, corresponding to Laporte-forbidden f–f transitions:12-Sm shows absorptions at λmax = 1370 nm (7300 cm–1), ε = 14 M–1 cm–1 and 1285 nm (7782 cm–1), ε = 13 M–1 cm–1, which arise due to 6H5/2 → 6FJ transitions; 2-Tm shows absorptions at λmax = 1549 nm (6456 cm–1), ε = 6 M–1 cm–1 and λmax = 1383 nm (7230 cm–1), ε = 15 M–1 cm–1 which arise due to ligand field-split 3H6 → 3H4 transitions; 2-Yb has a broad feature at λmax = 1015 nm (9552 cm–1), ε = 77 M–1 cm–1 and two weaker absorptions at λmax = 904 nm (11 061 cm–1), ε = 26 M–1 cm–1 and λmax = 844 nm (11 840 cm–1), ε = 27 M–1 cm–1 which correspond to 2F7/2 → 2F5/2 transitions, showing the ligand field splitting in the excited 2F5/2 term. These absorptions are moderately strong for f–f transitions because they are all spin-allowed (ε < 200 M–1 cm–1).2F7/2 → 2F5/2 manifold is a common feature for Yb3+ complexes; Da Re et al.et al.Dilute solutions of V region . Complex25 nm 23 0 cm–1, ε3-Sm, 3-Tm and 3-Yb are pale yellow, green and red, respectively, and as with 2-Ln their absorption spectra, are dominated by ligand to metal charge transfer bands tailing in from the UV region (3-Sm: λmax = 376 nm (26 595 cm–1), ε = 713 M–1 cm–1; 3-Tm: λmax = 327 nm (30 581 cm–1), ε = 378 M–1 cm–1; 3-Yb: λmax = 418 nm (23 923 cm–1), ε = 250 M–1 cm–1, λmax = 326 nm (30 674 cm–1), ε = 99 M–1 cm–1]. In the near IR region f–f absorptions are observed for all complexes; 3-Sm exhibits three main peaks at ν̃ 7246, 7710 and 8439 cm–1 due to 6H5/2 → 6FJ transitions, however there appear to be numerous weaker transitions. Complex 3-Tm shows two main absorptions at λmax = 1506 nm (6640 cm–1), ε = 47 M–1 cm–1 and λmax = 777 nm (12 870 cm–1), ε = 86 M–1 cm–1, corresponding to 3H6 → 3H4 and 3H6 → 3F4 transitions, however again these are structured due to ligand field splitting. Complex 3-Yb displays two absorptions at λmax = 973 nm (10 277 cm–1), ε = 22 M–1 cm–1 and λmax = 860 nm (11 627 cm–1), ε = 17 M–1 cm–1 corresponding to ligand field-split 2F7/2 → 2F5/2 transitions. The f–f transitions are at higher energy for 3-Ln, presumably due to stronger ligand fields; this is most clear for the Yb pair, where for 2-Yb the lowest energy transition is at 9500 cm–1, whilst this is seen at 10 200 cm–1 for 3-Yb.Solutions of V region   and it has easy-plane magnetic anisotropy as shown by EPR spectroscopy in both solid and frozen solution state with g1 = 5.6, g2 = 3.0 and g3 = 1.0.1-Tm give g1 = 5.49, g2 = 3.60 and g3 = 1.15, with g3 oriented along the N–Tm–N direction, resulting from a 99% pure |mJ| = 1/2 ground doublet.vs. the N–Ln–N angle, we performed further CASSCF-SO calculations on the structure of 1-Tm [N–Ln–N 166.89(6)°] where we substitute Yb3+ in place of Tm2+, and on the structure of 2-Yb [N–Yb–N 127.7(2)°] where we substitute Tm2+ in place of Yb3+ (note the change in ion charge to maintain an f13 configuration in both cases). We find the former to have an |mJ| = 1/2 ground doublet , and the latter to have an |mJ| = 7/2 ground doublet : thus, it is the structure that dictates these differing properties for f13 configurations and it is not due to the identity of the metal ion. Nocton and co-workers have recently made similar observations for isoelectronic f13 Tm2+ and Yb3+ 18-crown-6 complexes.Comparing 2-Yb we have carried out a systematic ab initio investigation. CASSCF-SO calculations have been performed on model structures based on the experimental structure of 2-Yb in which the N–Ln–N angle has been varied between 180° and 110°. The calculated g-values of the ground Kramers doublet of Yb3+ show a clear dependence of the type of magnetic anisotropy on the N–Ln–N angle, with the switching point located between 140° and 150°  for N–Ln–N angles <140° and easy-plane-like  for angles> 150°. This implies that there must be a significant structural change in the N–Yb–N angle of 2-Yb in the solution phase, becoming at least 150°. Optimization of the structure of 2-Yb in the gas phase using density-functional theory (DFT) shows an increase in the N–Yb–N angle from 127 to 133° °, the axis of quantization bisects the N–Tm–N angle and thus does not follow simple electron density arguments.We have conducted the same angular-dependent study of the electronic structure of 3+ ion in 2-Yb, we have performed a CASSCF-SO calculation on a model complex where the ligands have been trimmed to {N(SiH3)2}, thus removing the Ln···C and Ln···H electrostatic contacts; these calculations reveal changes of <10% to the SO energy levels (ESI Table S102-Yb. Although we cannot rule out changes to the N-donor strength for the trimmed ligand versus {N(SiiPr3)2}, these results suggest that the Ln···Cγ–Siβ agostic-type interactions have only a slight influence on the electronic structure at the Yb(iii) site and that they are far weaker than the Ln–N coordination bonds that dominate the electronic structure.To examine the impact of the Ln···Cγ–Siβ agostic-type interactions on the electronic structure of the Yb3+ complex, it is relevant to predict what the SMM properties of such a material could be now that we are far closer to a representative material with 2-Tm and 2-Yb, than in the previously reported near-linear 1-Sm.2-Tm where Tm3+ has been replaced with Dy3+. As predicted based on simple model compounds,ca. 1300–1400 cm–1 3}2]+ cations can be accredited to the formation of multiple electrostatic contacts between the highly Lewis acidic Ln3+ ions and the electron density associated with the ligand σ-bonding network, together with dipole stabilization, crystal packing forces and dispersion force interactions. By a combination of magnetic studies, EPR spectroscopy and ab initio calculations we have deduced the electronic structures of the bent Ln3+ cations. Interestingly, in the solid state [Yb{N(SiiPr3)3}2]+ expresses a similar crystal field to its three-coordinate precursor, rather than the axial crystal field that would be predicted for a two-coordinate complex. EPR spectroscopy shows that [Yb{N(SiiPr3)3}2]+ switches to an axial crystal field in solution, indicating that the N–Ln–N angle is less bent in the solution phase. The electronic structures of these bent Ln3+ cations are therefore sensitive to changes in molecular geometry.The preference for bent geometries in [Ln{N(Si3+ complexes such as [Dy{N(SiiPr3)2}2]+, proposed as SMMs with large energy magnetization reversal barriers,iPr3)2}2]+ cation is predicted to show a lower effective barrier to magnetic reversal than a linear analogue, it would be of benefit to be able to predict what ligand systems would provide two-coordinate Dy3+ complexes that are less bent. Although the [Ln{N(SiiPr3)2}2]+ framework is of sufficient steric bulk, a linear geometry is not enforced as the coordination sphere is flexible enough to be rearranged to increase the strength of ligand–metal electrostatic and ligand–ligand London dipole interactions. Given that recently isolated linear Dy2+ and Tb2+ metallocene systems have been proposed to exhibit significant s–d mixing,3+ complexes.Our synthetic results show that axial Dy2 and was stored over 4 Å molecular sieves. All solvents were degassed before use. For NMR spectroscopy C6D6 was dried by refluxing over K and CD2Cl2 was dried by refluxing over CaH2. Both NMR solvents were vacuum transferred and degassed by three freeze–pump–thaw cycles before use. 1-Ln,3)2][B(C6F5)4]3-SmAll manipulations were conducted under argon with the strict exclusion of oxygen and water by using Schlenk line and glove box techniques. Toluene, benzene and hexane were dried by refluxing over potassium and were stored over potassium mirrors. Dichloromethane (DCM) was dried over CaH1H (400 MHz), 13C{1H} (100 MHz and 125 MHz), 13C{19F} (126 MHz), 11B{1H} (128 MHz) and 19F{1H} (376 MHz) NMR spectra were obtained on an Avance III 400 MHz or 500 MHz spectrometer at 298 K. These were referenced to the solvent used, or to external TMS , H3BO3/D2O (11B) or C7H5F3/CDCl3 (19F). UV-vis-NIR spectroscopy was performed on samples in Youngs tap-appended 10 mm path length quartz cuvettes on an Agilent Technologies Cary Series UV-vis-NIR Spectrophotometer from 175–3300 nm. FTIR spectra were variously recorded as Nujol mulls in KBr discs on a PerkinElmer Spectrum RX1 spectrometer or as microcrystalline powders using a Bruker Tensor 27 ATR-Fourier Transform Infrared (ATR-FTIR) spectrometer. EPR spectroscopic measurements were performed at X-band using a Bruker super-high-Q X-band resonator attached to a Bruker EMX bridge, on solid state and frozen solution samples contained in flame-sealed quartz EPR tubes. Elemental analysis was carried out by Mr Martin Jennings and Mrs Anne Davies at the Microanalytical service, School of Chemistry, the University of Manchester. Elemental analysis results for 2-Yb reproducibly gave low carbon values; this has consistently been seen for {N(SiiPr3)2} complexes and we have previously attributed this observation to the formation of carbides from incomplete combustion.2-Ln and 3-Ln.3-Sm  and [H(SiEt3)2][B(C6F5)4]  and the resultant orange reaction mixture was stirred overnight at room temperature. The solvent was removed in vacuo and the oily red solid was washed with hexane (3 × 20 mL) and dried in vacuo for 1 h. The resultant red solid was cooled to –78 °C, dissolved in DCM (5 mL), layered with hexane (10 mL) and stored overnight at –25 °C to yield red crystals of 2-Sm . Anal. calcd (%) for C60H84N2Si4BF20Sm: C, 48.47; H, 5.69; N, 1.88; found: C, 47.25; H, 5.63; N, 1.72. χT product : 0.43 cm3 mol–1 K. 1H NMR : δ = –5.27 2), 0.43 2). 11B{1H} NMR : δ = –16.76 (s). 19F NMR : δ = –133.17 , –163.71 , –167.60 . The paramagnetism of 2-Sm precluded assignment of its 13C{1H} and 29Si NMR spectra. IR : 2954 (s), 2870 (s), 2813 (s), 1642 (s), 1511 (s), 1459 (s), 1384 (m), 1273 (s), 1082 (s), 978 (s), 928 (s), 881 (s), 765 (m), 693 (s), 676 (m), 543 (s), 489 (s), 415 (s) cm–1.Benzene (30 mL) was added to 3-Tm  and [H(SiEt3)2][B(C6F5)4]  and the resultant yellow reaction mixture was stirred overnight at room temperature. The solvent was removed in vacuo and the oily yellow-green solid was washed with hexane (3 × 20 mL) and dried in vacuo for 1 h. The resultant yellow-green solid was cooled to –78 °C, dissolved in DCM (5 mL), layered with hexane (10 mL) and stored overnight at –25 °C to yield yellow-green crystals of 2-Tm . Anal. calcd (%) for C60H84N2Si4BF20Tm: C, 46.06; H, 5.45; N, 1.76; found: C, 46.01; H, 5.55; N, 1.70. χT product : 6.44 cm3 mol–1 K. 1H NMR : δ = 25.04 2). 11B{1H} NMR : δ = –12.39 (s). 19F NMR : δ = –128.51 . The paramagnetism of 2-Tm precluded assignment of its 13C{1H} and 29Si NMR spectra. IR (Nujol): 2359 (m), 2340 (m), 1643 (w), 1514 (m), 980 (m), 918 (w), 897 (w), 800 (w), 773 (w), 756 (w), 700 (w), 683 (w), 667 (w), 660 (w) cm–1.Benzene (30 mL) was added to 3-Yb  and [H(SiEt3)2][B(C6F5)4] . The resultant dark purple reaction mixture was allowed to warm to room temperature slowly and stirred overnight. The solvent was removed in vacuo and the oily dark purple solid was washed with hexane (3 × 20 mL) and dried in vacuo for 1 h. The resultant dark purple solid was cooled to –78 °C, dissolved in DCM (1.5 mL), layered with hexane (3 mL) and stored at –35 °C overnight to yield dark purple crystals of 2-Yb . Anal. calcd (%) C60H84N2Si4F20BYb·CH2Cl2: C, 45.94; H, 5.44; N, 1.76; found: C, 44.81; H, 5.18; N, 1.58. χT product : 2.13 cm3 mol–1 K. 1H NMR : δ = 11.02 2). 11B{1H} NMR : δ = –14.67 (s). : δ = –131.58 , –162.05 , –165.15 . The paramagnetism of 2-Yb precluded assignment of its 13C{1H} and 29Si NMR spectra. IR (Nujol): 1267 (w), 1086 (m), 980 (m), 945 (w), 885 (w), 800 (w), 704 (m), 660 (m) cm–1.Toluene (15 mL) was added to a pre-cooled (–78 °C) mixture of tBuCl  in toluene (10 mL) was added dropwise to a pre-cooled (–78 °C) solution of 1-Tm . The reaction mixture was allowed to warm slowly to room temperature and was stirred at room temperature for 30 min, resulting in a colour change from dark brown to light brown. Volatiles were removed in vacuo and the product was extracted with hexane (10 mL), filtered, concentrated to 7 mL and stored at –35 °C overnight to yield pale green crystals of 3-Tm . Anal. calcd (%) C36H84N2Si4ClTm: C, 50.17; H, 9.82; N, 3.25; found: C, 50.39; H, 10.23; N, 4.11. χT product : 6.31 cm3 mol–1 K. The paramagnetism of 3-Tm precluded assignment of its 1H, 13C{1H} and 29Si NMR spectra. IR (Nujol): 1260 (w), 1245 (w), 1077 (w), 1061 (w), 1012 (m), 991 (w), 934 (s), 879 (m), 799 (w), 728 (m), 701 (s), 667 (m), 632 (m), 598 (m) cm–1.A solution of 1-Yb  and [Fe(Cp)2][PF6]  with stirring, and a white vapour was observed. The orange reaction mixture was stirred overnight at room temperature. All volatiles were removed in vacuo and ferrocene was sublimed away from the crude product at 90 °C for 1.5 hours. The remaining crude orange powder (1.029 g) was extracted with hexane (10 mL), filtered, concentrated to 7 mL and stored at –35 °C overnight to yield orange-red crystals of 3-Yb . Anal. calcd (%) C36H84N2Si4FYb·0.8C6H14: C, 53.36; H, 10.45; N, 3.05; found: C, 53.92; H, 10.87; N, 3.73. χT product : 1.78 cm3 mol–1 K. The paramagnetism of 3-Yb precluded assignment of its 1H, 13C{1H}, 19F and 29Si NMR spectra. IR (Nujol): 1247 (w), 1214 (w), 1071 (w), 1012 (w), 996 (w), 944 (m), 882 (m), 800 (w), 703 (m), 665 (m) cm–1.Toluene (20 mL) was added to a pre-cooled (–78 °C) mixture of There are no conflicts to declare.Supplementary informationClick here for additional data file.Video abstractClick here for additional data file.Crystal structure dataClick here for additional data file.


The title compound features a very short, but asymmetric, O—H⋯O hydrogen bond having an O⋯O distance of 2.452 (3) Å within the anion. 3,8]pentadeca-1(15),3(8),4,6,9,11,13-heptaen-10-yl]-1-methylpiperazin-1-ium 3,5-dinitrobenzoate–3,5-dinitrobenzic acid (1/1)}, C18H20ClN4+·C7H3N2O6−·C7H4N2O6, there is a very short, asymmetric, O—H⋯O hydrogen bond [O⋯O = 2.453 (3) Å] within the anion. The oxygen atoms of one of the nitro groups of the anion are disordered over two sets of sites having occupancies of 0.56 (3) and 0.44 (3). The fused tricyclic portion of the cation adopts a butterfly conformation, with a dihedral angle of 45.59 (6)° between the planes of the two aryl rings. In the crystal, a combination of O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds links the component species into a three-dimensional framework. Comparisons are made with the structures of some related compounds.In the title salt {systematic name: 4-[6-chloro-2,9-diazatricyclo[9.4.0.0 H-dibenzodiazepine, C18H19ClN4, is a well established medication for the treatment of schizophrenia, often preferred over other treatments because of the generally lower incidence of adverse side effects -5et al., 2015H-thioxanthen-9-yl)-N,N-dimethylpropan-1-amine , (I)i.e., the asymmetric unit contains one C18H20ClN4+ clozapinium cation and one C14H7N4O12− hydrogen bis anion −·(C7H4N2O6). The –CO2H and –CO2− groups in the anion are linked by a very short O22—H22A⋯O32 hydrogen bond ; H22A⋯O32 = 1.35 (4) Å]. There is a similarly short O—H⋯O hydrogen bond in the corresponding species of the chlorprothixene salt (III) on Fig. 1. An alted Table 1  and 0.44 (3) for the oxygen atoms. The major and minor disorder components make dihedral angles with the adjacent aryl ring of 17.2 (8) and 19.4 (8)°, with a dihedral angle between their own planes of 36.5 (14)°, so that these components are rotated out of the plane of the aryl ring in opposite senses.18H20ClN4+ cation of (I)In the Cet al., 1998abet al., 2000i =C(CN)COCMe3, of the 1,3 diketone 4-cyano 2,2,6,6-tetramethyl3,5-heptanedione, where the intramolecular O—H⋯O hydrogen bond has an O⋯O distance of 2.3936 (15) Å et al., 2016et al., 2018et al., 2015et al., 2015N-oxide, the corresponding angle is 56.2° 2O5. Crystals of (I)v/v); m.p. 494–497 K.Clozapine  and 3,5-dinitrobenzoic acid  were dissolved in methanol (10 ml), and this mixture was then stirred at 333 K for a few minutes. The solution was permitted to cool to room temperature and the resulting crystals were then collected by filtration and dried over P3) or 0.97 Å (CH2), and with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C); the CH3 group was permitted to rotate but not to tilt. For the H atoms bonded to N or O atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O). For the minor disorder component, the N—O distances and the 1,3-non-bonded O⋯O distances were restrained to be the same of the corresponding distances in the major component, subject to s.u. values of 0.01 and 0.02 Å, respectively. In addition, a similarity restraint was applied to the disordered O-atom sites and for each of the disorder components, the C—NO2 fragment was restrained to be planar. Subject to these conditions, the refined disorder occupancies are 0.56 (3) and 0.44 (3).Crystal data, data collection and refinement details are summarized in Table 210.1107/S2056989020012268/hb7944sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989020012268/hb7944Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020012268/hb7944Isup3.cmlSupporting information file. DOI: 2027224CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We found that negatively freshly isolated Vδ2+ T cells do not exhibit suppressive behavior, even after stimulation with IL-12/IL-18/IL-15 or the sheer contact with butyrophilin-3A1-expressing tumor cell lines (U251 or SK-Mel-28). On the other hand, Vδ2+ T cells positively isolated through TCR crosslinking or after prolonged stimulation with isopentenyl pyrophosphate (IPP) mediate strong inhibitory effects on αβ T cell proliferation. Stimulation with IPP in the presence of IL-15 induces the most robust suppressive phenotype of Vδ2+ T cells. This indicates that Vδ2+ T cells’ suppressive activity is dependent on a TCR signal and that the degree of suppression correlates with its strength. Vδ2+ T cell immunosuppression does not correlate with their Foxp3 expression but rather with their PD-L1 protein expression, evidenced by the massive reduction of suppressive activity when using a blocking antibody. In conclusion, pharmacologic stimulation of Vδ2+ T cells via the Vδ2 TCR for activation and expansion induces Vδ2+ T cells' potent killer activity while simultaneously licensing them to suppress αβ T cell responses. Taken together, the study is a further step to understand—in more detail—the suppressive activity of Vδ2+ γδ T cells.Despite recent progress in the understanding of γδ T cells’ roles and functions, their interaction with αβ T cells still remains to be elucidated. In this study, we sought to clarify what precisely endows peripheral Vδ2The online version of this article (10.1007/s00262-019-02469-8) contains supplementary material, which is available to authorized users. Because of γδ T cells’ distinctive features—potent antitumor effect and independence from MHC restriction—a special interest has been taken in the application of Vδ2+ T cells in cancer immunotherapy [+ T cells have resulted in limited clinical response in solid tumors thus far, even though many clinically unresponsive patients exhibited sustained Vδ2+ T-cell activation and proliferation [+ T cells from eliciting long-lasting antitumor effects in vivo needs to be elucidated. γδ T cells are known to inhibit or suppress the maturation and/or activation of other immune cells under certain conditions [T lymphocytes are divided into two subsets by their expression of T cell antigen receptors (TCRs): αβ T cells (combination of an α chain and a β chain) and γδ T cells (combination of a γ chain and a δ chain). γδ T cells constitute a major T cell population in the epithelial tissues but represent a rare  T cell population in the peripheral circulation with the majority (50–95%) of γδ T cells carrying a Vγ9Vδ2 TCR [otherapy . Howeverferation –13. Thernditions , 15. In nditions –18.+ T cell population has been identified in multiple types of solid cancers [+ T cells exert suppressive function on αβ T cells. Casetti et al. showed that up to 30% of Vδ2+ T cells express the Foxp3 transcription factor when they are activated by isopentyl pyrophosphate (IPP) in the presence of IL-15 and TGF-β. Foxp3+-enriched Vδ2+ T cells, but not positively freshly isolated Vδ2+ T cells, displayed regulatory/immunosuppressive activity on αβ T cells when co-cultured with autologous PBMCs in the presence of anti-CD3/anti-CD28 mAb [. [Staphylococcus aureus enterotoxin-stimulated CD4+CD25- T cells. On the other hand, γδ T cells initially activated by anti-CD3/anti-CD28 in the presence of TGF-β and IL-15 suppressed CD4+CD25- T cells although Foxp3 in γδ T cells was downregulated after transient expression. In contrast to Casetti’s paper, it was also reported that positively freshly isolated γδ T cells, which are Foxp3-negative, can potently suppress the in vitro proliferation of CD4+ T cells in the presence of anti-CD3/anti-CD28 mAb stimulation in the co-culture [+ T cells stimulated with IPP, but not negatively freshly isolated Vδ2+ T cells, can inhibit the proliferation of CD4+ and CD8+ αβ T cells reacting to strong recall antigens or allo-antigens. Combining these findings, Peters et al. [+ T cells cannot be detected with the Treg-specific 259D mAb [+ T cells to exhibit cell-contact dependent suppression.A tumor-infiltrating immunosuppressive Vδ2 cancers , 20; an  cancers . Yet, coCD28 mAb . In the 8 mAb [.  neither -culture , 23. In -culture  demonstrs et al. , 22 sugg259D mAb . Clarity259D mAb . Taken t+ T cells on autologous αβ T cells, specifically to address whether and how graded TCR stimulation and or cytokines control regulatory activities of Vδ2+ T cells. We examined the effect of proliferation inhibition and apoptosis induction mediated by negatively or positively freshly isolated Vδ2+ T cells obtained from healthy donors in comparison with those stimulated with IL-12/IL-18 (TCR bypass) + IL-15 and those after prolonged exposure to IPP with or without Th1 or Th2 cytokines. In addition, we tested the suppressive activity of Vδ2+ T cells in the presence or absence of a PD-1 blocking antibody. Next, to determine whether physiologic stimuli, such as the direct contact with tumor cells, affect the suppressive activity of Vδ2+ T cells, we exposed Vδ2+ T cells to a glioblastoma cell line (U251) or a melanoma cell line (SK-Mel-28) and subsequently examined these Vδ2+ T cells in mixed lymphocyte cultures (MLC) with anti-CD3/anti-CD28 stimulated autologous αβ T cells. Finally, we investigated the suppressive activity of Vδ2+ T cells in the presence or absence of CD28 stimulation. By employing Vδ2+ T cells that had experienced a range of stimuli, from none at all to supraphysiological levels, we identified conditions under which Vδ2+ T cells exerted suppressive effects on autologous αβ T cells.In the thymus, differences in signal strength dictate αβ versus γδ lineage choice through modulation of lineage specific transcription factors, while other signaling pathways that integrate with TCR signaling impact the resulting lineage outcome through altering activity of key proteins . In ligh+ T cells were isolated as follows: for positive isolation, PE-anti-Vδ2 antibody , anti-PE Microbeads , and MS columns (Miltenyi Biotec) were used; and for negative isolation, initially pan γδ T cells were isolated by using a custom-made gammadelta T cell isolation kit , and Vδ1+ T cells were subsequently depleted by using PE-anti-Vδ1 mAb , anti-PE MicroBeads, and LD columns (Miltenyi Biotec) according to the manufacturer’s protocol. CD3+ T cells were isolated through PE-anti-CD3 mAb , anti-PE Microbeads (Miltenyi Biotec), and MS columns (Miltenyi Biotec) according to the manufacturer’s protocol. Purity of Vδ2+ T cells was > 98% for positive isolation and > 99% for negative isolation. Purity of CD3+ T cells was always > 99%. Stimulation with IPP: according to Casetti et al. [6 cells/ml in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine , as well as IPP  and IL-2  in the presence or absence of IL-15 , TGF-β1  or IL-12 . On days 3, 6, and 9, half of the culture medium was replaced by fresh medium containing cytokines. TCR-bypass stimulation: negatively freshly isolated Vδ2+ cells were incubated for 24 h with IL-12 (50 ng/ml), IL-15 (50 ng/ml), and IL-18  in complete RPMI medium. Stimulation with anti-γδTCR mAb: culture plates were coated by overnight incubation with a TCRγδ mAb  at 4 ˚C. Then positively freshly isolated Vδ2+ cells were seeded in a flat bottom 96-well plate at 5×105 cells/200 µl/well and cultured overnight. Viability of both freshly isolated and cultured Vδ2+ T cells was examined routinely via flow cytometry by using the live/dead fixable violet dead cell stain kit  and were always > 99%. We summarize denominations and corresponding isolation methods in Table PBMCs from healthy donors were isolated by Ficoll density gradient centrifugation . Vδ2i et al. , PBMCs w+ T cells (1×105 cells) were cultured at a 1:1 cell ratio in a flat bottom 96-well plate containing complete RPMI medium and IL-2 (50 U/ml) with autologous PBMCs. When untouched or TCR-bypass-stimulated Vδ2+ T cells were analyzed, autologous freshly isolated PBMCs had been activated by anti-CD3/anti-CD28 Dynabeads for 48 h beforehand . In this case, anti-CD3/anti-CD28 Dynabeads were removed before starting MLC to strictly avoid the unintentional TCR stimulation on Vδ2+ T cells. When using positively freshly isolated Vδ2+ T cells from blood or IPP-stimulated PBMCs, anti-CD3/anti-CD28 Dynabeads were added in MLC simultaneously when starting co-culture with autologous PBMCs without prior stimulation. To disrupt the interactions between PD-1 and PD-L1, we coated plates in advance overnight at 4 °C with a blocking antibody to PD-1 (Pembrolizumab) or isotype control at a concentration of 5 μg/ml in designated experiments. To investigate the role of CD28 stimulation on Vδ2+ T cells for their suppressive activity, IMMU510-stimulated Vδ2+ T cells were cultured at a 1:1 cell ratio with autologous PBMCs or T cells in a flat bottom 96-well plate coated with either anti-CD3 mAb  or with anti-CD3 in combination with anti-CD28  mAb. Moreover, freshly isolated untouched Vδ2+ T cells were cultivated overnight in wells coated with anti-CD28 mAb , anti-TCRγδ mAb , or both antibodies and then cultured at a 1:1 cell ratio with autologous T cells on anti-CD3 antibody-coated  wells in a flat bottom 96-well plate.Isolated Vδ2+ cells and relative frequency of apoptotic cells was calculated by subtracting the frequency of annexin V+ cells in CD3/CD28-stimulated αβ T cells in the absence of Vδ2+ T cells.Cells were harvested and stained with PE-anti-Vδ2 (clone B6), APC-Cy7-anti-CD3 (clone SK7), both BD Bioscience, San Diego, USA; APC-anti-TCR αβ , followed by FITC-annexin V and 7AAD  24 h after MLC. Apoptotic cells were measured on a BD FACS CantoII flow cytometer and analyzed by using FlowJo Software v10 . Apoptotic cells were defined as annexin V+ T cells.The proliferation of αβ T cells in MLC was examined using the CellTraceTM Violet Cell Proliferation Kit (Thermo Fisher Scientific). To this end autologous PBMCs were labelled with CTV before MLC. On day 3 of MLC, cells were stained with APC-Cy7-anti-CD3, APC-anti-TCR αβ, 7AAD and FITC-anti-TCR γδ  mAbs. Proliferation of αβ T cells was measured on a BD FACS CantoII flow cytometer and analyzed by using FlowJo Software. CTVlow cells were calculated as proliferating cells. Inhibition of proliferating cells was calculated by comparing with the frequency of CTVlow cells in CD3/CD28-stimulated αβ T cells in the absence of Vδ2+ T cells with or without autologous PBMCs at 10 μl/ml and cells were incubated for 4 h at 37 °C with 10 μg/ml of brefeldin A (Biolegend) as well as 6 μg/ml of monensin . Rationale in brief: Monensin, which interacts with Golgi transmembrane transport, is the preferred choice when staining for only CD107a and not intracellular antibodies. Brefeldin A (BFA), which redistributes proteins from the Golgi to the ER, is commonly used when wanting to detect both CD107a and IFN. Both BFA and Monensin were used to detect CD107a and cytotoxins at the same time according to previous reports [APC-anti-PD-1 (clone EH12.2H7), PE-Cy7-anti-PD-L1 clone 29E2A3), APC-anti-CTLA4 (clone L3D10), APC-anti-perforin (clone B-D48), PE-anti-granzyme B , APC-Cy7-anti-CD3 (clone SK7), APC-anti-TCR αβ (clone BW242/412), 7AAD, FITC-anti-γδ TCR (clone 11F2), PE-anti-Vδ2 TCR antibody (clone B6), APC-H7-anti-CD28, FITC-anti-CD107a , and APC-anti-CD277  were used according to the manufacturer’s instructions. FITC-anti-CD107a antibody was added directly to the cell culture medium of Vδ2E2A3, APC reports . For intFor transcript quantification, RNA was extracted with the use of RNeasy Mini Kit  and reverse transcription was carried out using Superscript III First Strand Synthesis Super Mix  as described .For quantitative analysis, primers specific for Foxp3, PD-1, PD-L1, perforin, granzyme B, and GAPDH were used in real-time PCR with the SYBR Green kit  in a BioRad C1000 Thermal cycler/CFX96 real-time System . Briefly, 5 ng cDNA was added to a final volume of 10 µl/reaction containing 1 × SYBR Green PCR Master Mix  and 100 nM of each primer. Thermal cycling conditions were: denaturation at 95˚C 2 min, 42 cycles: 95 °C/10 s, 59 °C/15 s and 72 °C/30 s for elongation. Primers: GAPDH: forward 5′-CCACATCGCTCAGACACCAT-3′ and reverse 5′-GGCAACAATATCCACTTTACCAGACT-3′. Foxp3: forward 5′-GAGAAGCTGAGTGCCATGCA-3′ and reverse 5′-GGAGCCCTTGTCGGATGAT-3′. PD-1: forward 5′-ATCAAAGAGAGCCTGCGGG-3′ and reverse 5′-GGTGGGCTGTGGGCACT-3′. PD-L1: forward 5′-AAATGGAACCTGGCGAAAGC-3′ and reverse 5′-GATGAGCCCCTCAGGCATTT-3′. Granzyme B: forward 5′- TTCGTGCTGACAGCTGCTCACT-3′ and reverse 5′- CTCTCCAGCTGCAGTAGCATGA-3′. Perforin: forward 5′-ACCAGCAATGTGCATGTGTCTG-3′ and reverse 5′- GCCCTCTTGAAGTCAGGGT-3’.4 cells/ml in complete RPMI medium on day 0. On day 1, 6 × 105 isolated Vδ2+ T cells were added onto adherent tumor cells in a 24-well plate in complete RPMI medium supplemented with IL-2 (50 IU/ml). On day 3, Vδ2+ T cells were collected from the supernatant of culture medium for subsequent MLC.Adherent U251 (a glioblastoma cell line) or SK-Mel-28 (a melanoma cell line) cells were seeded in a 24-well plate at a cell concentration of 6 × 104 cells/well of the E-Plate in a volume of 100 µl and allowed to passively adhere on the electrode surface. Post seeding, the E-Plate was transferred to the RTCA MP instrument inside a cell culture incubator and incubated for the first 24 h without effector cells. Then, γδ T cells—untreated or treated with zoledronate—were applied onto the adherent tumor cells, with different effector cell: target cell ratios  in a volume of 100 µl. Changes in impedance were reported as Cell Index, which indicates attachment and adherence of cells to the plate’s electrode, every 30 min for the following 96 h as previously described [The xCELLigence RTCA MP instrument (ACEA Biosciences) was utilized for cytotoxicity assays. First, 50 µl of culture media was added to each well of 96 well E-Plates (ACEA Biosciences) and the background impedance was measured. Dissociated adherent target cells (U251 or SK-Mel-28) were seeded at a density of 1 × 10escribed . Data ant test. Comparisons between two groups were done using Student’s t test since both groups were normally distributed with equal variance. For comparisons among multiple groups, one-way ANOVA followed by Tukey’s multiple comparison test was used. The relationships between molecules expressed on γδ T cells and their suppressive activity were compared using Fisher’s exact test; the linear relationships were determined using Pearson's test. Statistical significance was defined as p < 0.05.Statistical tests were performed with GraphPad PRISM v8. Unpaired and parametric data with same SDs can be assessed by Student’s + T cells after four different magnitudes of TCR stimulation, namely negatively freshly isolated Vδ2+ T cells (no stimulation), freshly untouched isolated Vδ2+ T cells with subsequent TCR-bypass stimulation (TCR-independent cytokine-mediated stimulation), fresh positively isolated Vδ2+ T cells (a single strong γδ TCR stimulus), and IPP-stimulated Vδ2+ T cells (strongest and continuous γδ TCR stimulation) for their suppressive activity by co-culturing them with anti-CD3/CD28 activated autologous αβ T cells. Vδ2+ T cells activated in a TCR-independent manner by inflammatory cytokines (TCR-bypass stimulation) [+ T cells during positive selection, resulting in one strong TCR stimulus. Activation by PAgs is currently the basis of two main strategies involving Vδ2+ T cells for cancer immunotherapy [+ T cells by PAgs. Therefore, it is of critical importance to know whether PAg-stimulated Vδ2+ T cells have suppressive function or not. Thus γδ T cells stimulated for 10 days with IPP and subsequent positive isolation via the TCR using MACS technology represented the maximum TCR stimulatory manipulation in our model system. Since IL-15 and IL-12 alone or in combination enhanced the activation of PAg-stimulated γδ T cells [+ T cells, which then suppressed the proliferation of αβ T cells [To understand the role of the γδ TCR in determining functional differentiation into a regulatory phenotype, we examined peripheral Vδ2ulation) –36 mimicotherapy : (1) in  T cells , 36, 38  T cells , TCR sti+ T cells did not exhibit any inhibitory effect on the proliferation of anti-CD3/anti-CD28 stimulated αβ T cells, while TCR-bypass stimulation with IL-12/IL-18 [+ T cells positively isolated through an anti-TCR Vδ2 antibody, however, significantly suppressed the proliferation of autologous αβ T cells . We found mRNA expression of granzyme B to be significantly higher on TCR-bypass-stimulated Vδ2+ T cells compared to untouched IL-2 stimulated Vδ2+ T cells; however, the reverse was true for perforin expression. In fact, TCR bypass stimulation induced significantly more granzyme B than any other condition tested. None of the positively selected Vδ2+ T cells expressed appreciable amounts of granzyme B or perforin mRNA. Accordingly, there was no significant correlation between mRNA levels of cytotoxic granules and apoptosis induction . Then we investigated the protein expression of granzyme B, perforin, and CD107a on Vδ2+ T cells by flow cytometry after expansion with PAgs before adding them into MLC . During the process of cell killing, vesicles produced in an effector cell fuse with the target cell membrane, releasing cytotoxins such as perforin and granzymes. CD107a is a vesicle membrane protein that becomes transiently mobilized to the effector cell surface during this degranulation process [+ T cells after MLC with αβ T cells , indicating degranulation of perforin upon cell contact with target cells. Interestingly, granzyme B expression remained unchanged. In conclusion, although only low levels of apoptosis are induced (below 10%), expression levels of granzyme B, perforin, and CD107a correlate exactly with these levels, suggesting apoptosis induction by Vδ2+ T cells is dependent on cytotoxic granules; however, this constitutes only a very small part of the overall regulatory activity of Vδ2+ T cells.To identify a potential correlation between suppression and apoptosis induction, we examined the expression of cytotoxic granules by Vδ2 process . The use process –45. In c+ T cells is associated with their suppressive activity, we investigated the mRNA level of Foxp3 on Vδ2+ T cells. As shown previously [+ T cells expressed higher levels of Foxp3 in the presence of TGF-β and IL-15 compared to most other conditions  at a concentration of 5 μg/ml. While the apoptosis induction effect of IPP/IL-15- or IPP/IL-15/IL-12-stimulated Vδ2+ T cells was not altered , the proliferation inhibition effect of IPP/IL-15-stimulated Vδ2+ T cells was abrogated by 50% by the anti-PD-1 antibody  proteins are essential for human γδ T cell activation by PAgs . As show+ T cells with high CD80/86 expression are potent antigen presenting cells to αβ T cells [+ T cells, we first examined the suppressive activity of TCR-stimulated Vδ2+ T cells that were cultured in the absence of antigen presenting cells and CD28 stimulation prior to MLC  and stimulated with immobilized anti-TCRγδ antibody  for 24 h, and only then co-cultured with autologous PBMCs or CD3+ T cells on anti-CD3- or anti-CD3/anti-CD28-coated wells. While anti-CD28 mAb can be either immobilized or added soluble, we chose immobilized anti-CD28 mAb for reasons of comparison with anti-CD3/anti-CD28 bead stimulation [+ T cells—that had been positively selected and subsequently activated via immobilized IMMU510—with isolated CD3+ T cells, and compared these to those co-cultured with PBMCs  were used as targets, suggesting that CD28 stimulation may not be necessary for Vδ2+ T cells themselves to differentiate into a suppressive phenotype, yet CD28 stimulation may be relevant for the targets that get suppressed. Figure + T cells inhibited the proliferation of αβ T cells in PBMCs by 65.4% and those in pure T cells by 44.6%  and CD14 negative, thus unlikely monocytes, Supplementary Figure S8) cultivated in wells immobilized with anti-CD28, or anti-TCRγδ or both mAbs and then co-cultured them with autologous T cells stimulated with anti-CD3 mAb (OKT3). Different to in vivo situation where T cell proliferation is dependent on a second signal T cell proliferation solely by OKT3 was already proven in vitro in previous literatures in different settings [+ T cells exerted no suppressive effect. Accordingly, TCRγδ-stimulated Vδ2+ T cells inhibited the proliferation of αβ T cells to a similar level in the absence or presence of CD28-stimulation . These results indicate that CD28 stimulation does not induce additional suppressive activity on either untouched or TCR-stimulated Vδ2+ T cells.To further investigate the role of CD28 stimulation with respect to the induction of suppressive function of Vδ2settings –54. Figu+ T cells and even the adoptive transfer of allogeneic γδ T cells shows objective effects, complete remissions still remain anecdotical and the necessary repetitive in vivo activation shows limited responses [+ T cells from exerting long-lasting antitumor effects in vivo. Current in vitro and in vivo strategies employ pharmacological drugs targeting the Vδ2+ TCR for the induction of activation, proliferation, and tumor-lytic function of γδ T cells. By taking a closer look at the role of the TCR in this process, we demonstrate that the suppressive activity of Vδ2+ T cells on autologous αβ T cells correlates with the strength of γδ TCR signal and that the functional differentiation of γδ T cells into a suppressive phenotype is fine-tuned by the integration of signals arising from other environmental cues, such as cytokines. More specifically, negatively freshly isolated Vδ2+ T cells, TCR-bypass cytokine-stimulated negatively isolated Vδ2+ T cells, TCR-crosslinked freshly isolated Vδ2+ T cells, and IPP-stimulated TCR-crosslinked Vδ2+ T cells exhibit increasing inhibitory activities on αβ T cell proliferation, from the weakest to the strongest stimulation setting in this order. In contrast, direct contact with BTN3A1-expressing cells in vitro alone is not a sufficiently strong γδ TCR-stimulation to bestow suppressive activity on Vδ2+ T cells.Tumor-infiltrating γδ T cells have been considered the most favorable prognostic marker in many types of cancers . While tesponses . The pre+ T cells with four different magnitudes of TCR stimulation. First, we showed that negatively freshly isolated Vδ2+ T cells (no stimulation) do not possess suppressive activity in accordance with Traxlmayr and colleagues’ findings [+ Vδ2+ T cells because this fraction consists of 10–40% of peripheral Vδ2+ T cells in healthy donors [+ T cells to specifically analyze Vδ2+ T cells. Therefore, we believe that we studied the most purified (> 99%) untouched Vδ2+ T cells. Next, we found that TCR-bypass stimulation (TCR-independent cytokine-mediated stimulation) does not confer significant immunosuppressive function to negatively freshly isolated Vδ2+ T cells. To our knowledge, this is the first study to test the impact of TCR-bypass stimulation on the suppressive effect of truly untouched Vδ2+ T cells. Third, our study demonstrates that positively freshly isolated Vδ2+ T cells (a single γδ TCR stimulation) potently suppress the proliferation of co-cultured autologous αβ T cells that have been stimulated with anti-CD3/anti-CD28 mAb in whole PBMCs. Casetti et al. [+ T cells do not suppress the proliferation of anti-CD3/anti-CD28 mAb-stimulated PBMCs, while Peters et al. and Kuhl et al. showed that positively freshly isolated pan γδ T cells suppress the proliferation of CD4+ T cells in response to anti-CD3/anti-CD28 mAb stimulation [+ T cells. Finally, we reveal that IPP-stimulated Vδ2+ T cells (strongest and continuous γδ TCR stimulation) exhibit the most robust suppressive activity. Intriguingly, untouched Vδ2+ T cells do not suppress autologous αβ T cells even after exposure to BTN3A1+ tumor cells. As tumor-infiltrating γδ T cells are thought to be the most favorable prognostic marker in many types of cancers [+ T cells via pharmacologic drugs for activation and expansion may dampen the anti-tumor reactivity of αβ T cells indirectly by reducing their number, which points to a thus far unappreciated limitation to adoptive Vδ2+ T cell therapy for cancer patients. However, since T cells with high capacity of proliferation and those with high potential of cytokine-production or anti-tumor cytolytic function are different populations, we cannot conclude whether γδ T cells dampen αβ T cell responses per se. They may actually enhance the cytolytic potential of suppressed αβ T cells, which we did not examine in this study.We examined peripheral Vδ2findings . While ty donors . In addii et al.  concludemulation , 23. The cancers , this fi+ T cells yet very slightly increases apoptosis induction by Vδ2+ T cells  activated γδ T cells express CD28 [+ T cells after TCR signaling, i.e., in this study after IPP/IL-15-stimulation, and also the only marginal expression of CTLA-4 before and after IPP/IL-15-stimulation . In contrast, PD-1 and PD-L1 expression were significantly upregulated by the IPP-containing regimen. By using blocking antibodies, Peters et al. showed that CD28-CTLA-4/CD80-CD86 and PD-1-PD-L1 interactions are involved in the suppressive activity of Vδ2+ γδ T cells. Our experiments clearly confirm a role for the PD-1/PD-L1 axis. This finding also supports the notion that the TCR is decisive in inducing regulatory function in γδ T cells, as PD-L1 is positively regulated by TCR stimulation. We also demonstrate that CD28 stimulation on Vδ2+ T cells is not essential for Vδ2+ T cells to acquire suppressive function on autologous αβ T cells. However, since PD-1/PD-L1 interaction explains only about 50% of suppressive function of Vδ2+ T cells, CD28 stimulation on αβ T cells by suppressive Vδ2+ T cells is of relevance and needs to be elucidated as well as the role of other inhibitory receptors such as Tim-3, Lag-3 or TIGIT.Thus, although we and others have identified an array of conditions that create a suppressive phenotype in Vδ2ess CD28 –65. CTLAess CD28 . Our dat+ T cells was observed in the present study. On the contrary, the population with the least suppressive activity among PAg-stimulated Vδ2+ T cells (IPP/IL-15/IL-12 stimulation) had the highest Foxp3 expression and vice versa (IPP/IL-15 stimulation). This suggests that Foxp3 is a transient activation marker rather than a regulatory marker analogous to Foxp3 expression in activated human CD4 αβ T helper cells [+ T cells. This supports the finding that the interaction between PD-1 on αβ T cells and its ligand PD-L1 on γδ T cells restrains αβ T cell activation [+ T cells can be a marker to predict their suppressive activity and respective blocking antibodies massively reduced inhibitory function, this prediction is not applicable to TCR-bypass-stimulated Vδ2+ T cells, which express high PD-L1 but exhibit no suppression, or positively freshly isolated Vδ2+ T cells, which have only marginal expression of PD-L1 but possess high suppressive activity. On the other hand, it is already known that activated γδ T cells express CD80/86 [Similar to what is shown by Peters et al. , no correr cells , 67. Instivation . Interestivation  or by cotivation , but it  CD80/86 . Althoug+ T cells, like untouched and TCR-bypass-stimulated Vδ2+ T cells, did not exhibit any suppressive behavior, although BTN3A1 is suggested to be the natural Vδ2 TCR ligand, illuminates our incomplete understanding of what truly activates γδ T cells and those factors that contribute to Vδ2+ TCR signaling and thus (suppressive) effector differentiation in vivo. If assuming suppression resulting from crosslinking the TCR is 100%, IL-15 was able to significantly increase suppressive behavior further by 35% while other cytokines, such as TGF-β and IL-12, down-modulated inhibitory activity distinctively. This denotes a decisive role of environmental cues in (suppressive) effector differentiation of γδ T cells. The excellent performance of Vδ2+ T cells in terms of viability throughout the experimental procedures and the consistently distinct modulation of the TCR signal by cytokine stimulation strongly suggest that these TCR signals represent a physiological range of activation and function. Until we have identified the physiological (co-)ligand(s) and factors that contribute to Vδ2+ T-cell activation, we are unable to delineate what specifically promotes suppressive γδ T cells in vivo.The finding that BTN3A1-exposed Vδ2+ T cells on αβ T cells is dependent on a TCR signal and correlates with its strength Below is the link to the electronic supplementary material.


Their structures with absolute configurations were elucidated by combination of spectroscopic analysis, single crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compound 1 was a rearranged isobutylphloroglucinol–pinene adduct featuring an unusual 2,3,4,4a,10,11-hexahydro-1H-3,11a-methanodibenzooxepin backbone. Compound 4 showed moderate antibacterial activity against Gram-positive bacteria including multiresistant strains.Myrcauones A–D (The online version of this article (10.1007/s13659-020-00288-4) contains supplementary material, which is available to authorized users. Myrciaria cauliflora is an evergreen shrub and widely distributed in southern and central Brazil oxepin backbone. All isolates were evaluated for their antibacterial activities. Herein, we describe the isolation, structural elucidation, and antibacterial activities of these myrcauones A–D (1–4).The plant l Brazil . This pll Brazil , 3. Prevl Brazil –6. As a l Brazil –10, four1 was obtained as yellow gum. The molecular formula of 1 was established as C22H32O3 by its HRESIMS data . The UV spectrum displayed absorption maximum at 206 nm. The IR spectrum showed characteristic absorptions for hydroxyl group (3475 cm−1) and aromatic ring (1611 and 1488 cm−1). The 1H NMR spectrum of 1 suggested the presence of an olefinic proton [δH 6.23 ], a hydroxyl group [δH 4.75 ], a methoxy group [δH 3.76 ], an isopropyl moiety , and three tertiary methyls . The 13C NMR and DEPT spectra of 1 exhibited 22 carbon signals including 7 quaternary carbons (5 olefinic ones), 5 methines (an oxygenated and an olefinic ones), 4 methylenes, and 6 methyls (an oxygenated one). The aforementioned data implied that 1 could be an isobutylphloroglucinol–monoterpene adduct , which was further confirmed by the two spin systems (H-2 to H-6 and H-10 to H-9′/H-10′) in its 1H–1H COSY spectrum . Furthermore, the HMBC correlations between H-7′ and C-1, and between H2-10 and C-1′ defined the connection of 1a and 1b via C-7′–C-10 bond. Finally, the leftover oxygen atom was anticipated to connect C-2 (δC 88.6) with C-6′ (δC 159.4) to form an uncommon 2,3,4,5-tetrahydrooxepine ring on the basis of the HMBC correlation between H-2 and C-6′ as well as the molecular formula information.A comparison of the NMR data of cadine A  indicate1 was established by a NOESY experiment  were β-oriented. Meanwhile, the correlations between H-10α and H-6a/H-7′ as well as between H-6a and Me-9 suggested that Me-9 and H-7′ were α-oriented. To determine the absolute configurations of 1, a comparison of its experimental and calculated ECD data was performed. The experimental ECD spectrum of 1 exhibited negative Cotton effects at 211 (Δε + 6.2) and 277 (Δε + 0.7) nm, and a negative one at 238 (Δε − 0.5) nm, which were similar with those in the calculated CD spectrum for 1R,2R,4S,7′S-isomer . The IR spectrum showed absorptions of hydroxyl (3357 cm−1), carbonyl group (1656 cm−1), and double bonds (1605 and 1462 cm−1). The 1H NMR data ], a methoxy group [δH 3.75 ], an isopropyl moiety , and three tertiary methyls  indicated that 2 could be an isobutylphloroglucinol–monoterpene adduct  , S. aureus ATCC 700699 (VISA), S. aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 and Gram-negative strains Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were measured by broth microdilution method. As a result, compound 4 exhibited moderate antibacterial activity against all Gram-positive strains with MIC value of 32 μg/mL . X-ray crystallographic analyses were carried out on an Agilent Gemini S Ultra CCD diffractometer with Cu Kα radiation (λ = 1.54178 Å). Silica gel , Sephadex LH-20 , and reversed-phase C18 silica gel  were used for column chromatography (CC). Preparative HPLC was carried out on an Agilent 1260 Chromatograph equipped with a G1311C pump and a G1315D photodiode array detector  with a semi-preparative C18 reversed-phase column . All solvents used in CC and HPLC were of analytical grade  and chromatographic grade , respectively.Melting points were obtained on a Buchi melting point B-545 apparatus  and are uncorrected. Optical rotations were measured on a JASCO P-2000 digital polarimeter  at room temperature. IR spectra were determined on a JASCO FT/IR-4600 plus Fourier transform infrared spectrometer  using KBr pellets. UV spectra were recorded on a JASCO V-550 UV/Vis spectrophotometer . CD spectra were obtained on a ChirascanqCD . HRESIMS spectra were acquired on an Agilent 6210 LC/MSD TOF mass spectrometer . NMR spectra were measured on Bruker AV-500 or AV-400 spectrometers  with TMS as internal standard, and chemical shifts were denoted in M. cauliflora were collected from Nanning city, Guangxi Province of People’s Republic of China, in July of 2018. A voucher specimen (No. 2018070607) identified by Professor Guang-Xiong Zhou (Jinan University) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, People’s Republic of China.The leaves of M. cauliflora (15 kg) were powdered and extracted with 95% EtOH  at room temperature. The extract (2.2 kg) was suspended in H2O and extracted with petroleum ether . The PE extract (673.2 g) was subjected to a silica gel column chromatography using cyclohexane–EtOAc  as eluent to afford 10 fractions (Frs. A–J). Fr. G (48.3 g) was further separated by silica gel column using a gradient cyclohexane–EtOAc  to give 8 subfractions (Frs. G1–G8). Subfraction G5 (10.7 g) was chromatographed on Sephadex LH-20  to obtain three subfractions (Frs. G5A–G5C). Subfraction G5B (7.3 g) was subjected to ODS column using MeOH/H2O  and further purified by semi-preparative reversed-phase HPLC  to afford 1 , 3  and 4 . Subfraction G6 (5.3 g) was separated on Sephadex LH-20  to obtain 2 (7.3 mg).The air-dried leaves of Compound 1 yellow gum (CH3OH); [α]D25 =  + 119 ; UV (MeOH) λmax (log ε) 206 (3.73) nm; IR (KBr) νmax 3475, 2977, 2954, 2876, 1611, 1584, 1488, 1445, 1415, 1385, 1307, 1236, 1199, 1132, 1084, 1035, 1014, 982, 904, 831 cm−1; 1H NMR  and 13C NMR , see Table m/z 345.2423 [M+H]+ ; ECD  211 (+ 6.2), 238 (− 0.5), 277 (+ 0.7) nm.Compound 2 yellow oil (CH3OH); [α]D25 =  + 59 ; UV (MeOH) λmax (log ε) 202 (3.85), 245 (3.92), 296 (3.50) nm; IR (KBr) νmax 3357, 2957, 2933, 2871, 1656, 1605, 1462, 1385, 1353, 1236, 1137, 1092, 998, 983, 893, 843 cm−1; 1H NMR  and 13C NMR , see Table m/z 361.2386 [M+H]+ ; ECD  203 (− 19.8), 244 (+ 11.7), 296 (+ 8.1), 337 (− 1.8) nm.Compound 3 yellow oil (CH3OH); [α]D25 =  − 63 ; UV (MeOH) λmax (log ε) 202 (3.71), 245 (3.77), 297 (3.32) nm; IR (KBr) νmax 3359, 2958, 2924, 2871, 1656, 1603, 1463, 1388, 1372, 1259, 1232, 1136, 1089, 989, 899, 842 cm−1; 1H NMR  and 13C NMR , see Table m/z 361.2391 [M+H]+ ; ECD  203 (+ 18.0), 245 (− 20.5), 293 (− 9.6), 333 (+ 2.5) nm.) 202 3.7, 245 (3.Compound 4 colorless blocks (CH3OH); m.p. 162–163 ℃; [α]D25 =  − 42 ; UV (MeOH) λmax (log ε) 205 (3.86), 247 (3.87), 310 (3.44) nm; IR (KBr) νmax 3336, 2957, 2870, 1663, 1615, 1462, 1386, 1364, 1284, 1259, 1233, 1176, 1140, 999, 941, 886, 844 cm−1; 1H NMR  and 13C NMR , see Table m/z 429.2996 [M + H]+ .Crystal data for 4 C27H40O4, monoclinic, space group P21, a = 10.2148 (3) Å, b = 24.2552 (4) Å, c = 20.4139 (5) Å, α = 90°, β = 97.254 (2)°, γ = 90°, V = 5017.3 (2) Å3, T = 293 (2) K, Z = 2, Dcalcd = 1.135 g cm−3, F(000) = 1872, 20761 reflections measured (2.18° ≤ θ ≤ 73.86°), 13408 unique  which were used in all calculations. The final R1 was 0.0916 [I > 2σ(I)] and wR2 was 0.2884 . CCDC-1998470 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.Staphylococcus aureus ATCC 43300 , S. aureus ATCC 700699 , S. aureus ATCC 25923, E. faecalis ATCC 29212, P. aeruginosa ATCC 27853, E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were standard isolates from ATCC . The MIC values were measured using a previously reported method [(PDF 2585 kb)


Psen1 KO mice and knock-in (KI) mice with homozygous FAD-associated L435F mutations (LF/LFPsen1) are embryonic and perinatally lethal, precluding a more rigorous examination of the effect of Alzheimer's disease–causing Psen1 mutations on neurodegeneration. Given that the rat is a more suitable model organism with regard to surgical interventions and behavioral testing, we generated a rat KI model of the LFPsen1 mutation. In this study, we focused on young LFPsen1 rats to determine potential early pathogenic changes caused by this mutation. We found that, unlike LF/LFPsen1 mice, LF/LFPsen1 rats survive into adulthood despite loss of γ-secretase activity. Consistent with loss of γ-secretase function, LF/LFPsen1 rats exhibited low levels of Aβ38, Aβ40, and Aβ42 peptides. In contrast, levels of Aβ43, a longer and potentially more amyloidogenic Aβ form, were significantly increased in LF/LFPsen1 and LF/wPsen1 rats. The longer survival of these KI rats affords the opportunity to examine the effect of homozygous Psen1 Alzheimer's disease–associated mutations on neurodegeneration in older animals.Familial forms of Alzheimer's disease (FAD) are caused by mutations in the gene encoding amyloid precursor protein, whose processing can result in formation of β-amyloid (Aβ). FAD can also result from mutations in the  PSEN1 and PSEN2, with the majority occurring in PSEN1 (PSEN1 (RRID: SCR_006416), and the biochemical effect of PSEN1 mutations is complex. In general, PSEN1 mutations result in a decrease in endopeptidase activity and altered γ-processivity, resulting in reduced amounts of γ-secretase products and a relative increase in the longer forms of γ-secretase products. With regard to amyloid precursor protein (APP) processing, PSEN1 mutations show reduced levels of β-amyloid (Aβ) . A CRISPR/Cas9-mediated knock-in system was chosen to avoid the artifacts induced by the transgenic approach . The rats were placed on a humanized APP background in PSEN1 . FAD-cauoid (Aβ) , whose aoid (Aβ)  and in vo models  of the FD brains  and cellD brains , 10 exprD brains , undetecy lethal  in a man KO mice , likely from PS2 . Analysihenotype . Here weckground  to accomLFPsen1 rats were crossed to Long-Evans rats to generate F1-LF/wPsen1 rats. These crossings were repeated four more times to obtain F5-LF/wPsen1 rats. The probability that F5 rats carry unidentified off-target mutations  is ∼1.5625%. To generate LFPsen1 rats on a background where rat App has a humanized Aβ region, F5-LF/wPsen1 and h/hApp rats were crossed to generate F1-LF/wPsen1; h/wApp rats. The wApp allele was removed in subsequent crosses. For all data generated in this study, all rats were on the h/hApp background, which produces human and not rodent Aβ species.F0-LFPsen1 mutations were correctly inserted into Psen1 exon 12, we amplified, by PCR, Psen1 gene exon 12 from w/wPsen1, LF/wPsen1, and LF/LFPsen1 rats. Sequencing of the PCR products showed that the mutations were correctly inserted in the LF/wPsen1 and LF/LFPsen1 genomes (A). RT-PCR analysis performed on RNA from P0 rat brain lysate from w/wPsen1, LF/wPsen1, and LF/LFPsen1 rats confirmed that no alterations in Psen1 or Psen2 expression were caused by the L435F mutation (B and C)  = 1.864, p = 0.1824; Psen2: F = 0.6833, p = 0.5169). KI LF/LFPsen1 mice display perinatal lethality . At the time of weaning (P27), male and female LF/LFPsen1 rats weighed significantly less than w/wPsen1 and LF/wPsen1 littermates (E)  = 30.53, p < 0.0001; post hoc Tukey's multiple comparisons test results are reported in the figures). LF/LFPsen1 rat survival declined to 65% by day 28 and then stabilized into adulthood, whereas w/wPsen1 and LF/wPsen1 littermates showed no significant postnatal lethality in the same period (F).To verify that the  genomes A. RT-PCRmutation , B and Cethality  and deve KO mice . To deten ratios D. At thetermates E   = 13.43, p = 0.0020; APP-αCTF M: F = 18.03, p = 0.0007; APP-βCTF F: F = 10.78, p = 0.0041; APP-βCTF M: F = 16.17, p = 0.0010; N-cad–CTF F: F = 7.415, p = 0.0125; N-cad–CTF M: F = 5.597, p = 0.0263; post hoc Tukey's multiple comparisons test results are reported in the figures). Full-length APP and N-cadherin were not significantly changed (A)  = 0.8284, p = 0.4675; mature APP M: F = 0.7954, p = 0.4807; immature APP F: F = 0.2181, p = 0.8082; immature APP M: F = 0.0478, p = 0.9534; N-cad–FL F: F = 0.5231, p = 0.6096; N-cad–FL M: F = 3.778, p = 0.0644). Analysis of PS1 showed a sex-independent and LFPsen1 allele dose-dependent decrease in autocatalysis of PS1 (i.e. Presenilinase activity), a prerequisite for γ-secretase function (LF/wPsen1 and LF/LFPsen1 rat brains (B)  F: F = 21.48, p = 0.0004; PS1-FL  M: F = 55.19, p < 0.0001; PS1-CTF F: F = 7.236, p = 0.0134; PS1-CTF M: F = 50.85, p < 0.0001; PS1-FL  F: F = 10.27, p = 0.0048; PS1-FL  M: F = 10.36, p = 0.0046; post hoc Tukey's multiple comparisons test results are reported in the figures). PS2 was not affected in a similar manner, nor were there any alterations in the levels of other members of the γ-secretase complex, Nicastrin and Pen2 (C)  = 3.135, p = 0.0926; PS2-CTF M: F = 0.2981, p = 0.7493; Nicastrin F: F = 3.360, p = 0.0813; Nicastrin M: F = 0.8258, p = 0.4685; Pen2 F: F = 1.497, p = 0.2746; Pen2 M: F = 1.193, p = 0.71920).As noted above, γ-secretase has several substrates, including APP and N-cadherin . Typicalcontrols A . LF/LFPsen1 rat brains had lower levels of Aβ38, Aβ40, and Aβ42 in a sex-independent manner (A)  = 59.20, p < 0.0001; Aβ40: F = 183.8, p < 0.0001; Aβ42: F = 41.91; post hoc Tukey's multiple comparisons test results are reported in the figures). LF/wPsen1 animals in general had similar amyloid levels compared with WT rats, with the exception of lower levels of Aβ38 in female LF/wPsen1 rats (A). Notably, the Aβ42/Aβ40 ratio was also increased in LF/LFPsen1 rat brain lysates in a sex-independent manner (A)  = 49.06, p < 0.0001). Overall, the decrease in γ-secretase products (Aβ peptides), increase in γ-secretase substrates , and decrease in the autocatalysis of PS1 in LFPsen1 rats is indicative of loss of γ-secretase function.Cleavage of APP-βCTFs by γ-secretase generates Aβ peptides, which vary in length depending on the processivity of γ-secretase; reduced processivity increases the relative amounts of longer Aβ peptides compared with shorter Aβ peptides. To complete the assessment of γ-secretase function in hemistry E. Psen1Lt manner A   = 8.119, p = 0.00004). We also detected an increase in relative levels of Aβ43/Aβ40 and Aβ43/Aβ42 ratios (B) Interestingly, only one allele with the LFPsen1 mutation was sufficient to drive the increase in Aβ43 levels, whereas the Aβ43/Aβ40 and Aβ43/Aβ42 ratios showed a trend of gene dose dependence and significant gene dose dependence, respectively  = 26.70, p < 0.0001; Aβ43/Aβ42: F = 15.15, p < 0.0001). Thus, the L435F mutation also reduces γ-secretase processivity in vivo, as indicated by the absolute (Aβ43) and relative (Aβ42) increases in longer Aβ peptides.Although Aβ40 and Aβ42 levels were decreased in d female B . Surprisingly, LF/LFPsen1 rat brain lysates show an increase in sAPPα levels and a decrease in sAPPβ levels (B) (sAPPα: F (2. 39) = 7.513, p = 0.0017; sAPPβ: F (2. 39) = 8.909, p = 0.0007), indicative of a secondary increase in α-secretase and decrease in β-secretase activity caused by the pathogenic Psen1 mutation.To assess α- and β-secretase cleavage of APP, solubilized brain lysates were analyzed by ELISA for soluble APP ectodomain levels, sAPPα and sAPPβ, the other products of α- and β-cleavage of APP, respectively. No differences were seen in  WT rats B. Surpriβ levels B , CASP8 and FADD-like apoptosis regulator (Cflar), and hairy and enhancer of split 1 and 5  levels in RNA derived from P0 w/wPsen1 and LF/LFPsen1 rat brain lysates showed no significant differences (A–D) . This result suggests that Notch signaling is not impaired in LF/LFPsen1 rats.Impaired neurogenesis in ferences , A–D  analysis to characterize brains from p15 w/wPsen1, LF/wPsen1, and LF/LFPsen1 rats. Regions of analysis included the frontal cortex, cingulate cortex, whole hippocampus, and entorhinal cortex. No gross morphological changes were evident by H&E staining in any of the rats analyzed (A). Qualitative inspection of NeuN staining showed no appreciable changes in neuronal density in any of the regions analyzed in LFPsen1 rats, but a quantitative analysis of total NeuN signal found a small but statistically significant increase in male LF/LFPsen1 rat whole hippocampus and the CA2–CA3 region (B). No evidence of astrocytosis or microgliosis was seen by staining with Glial fibrillary acidic protein and IBA1, respectively, in any of that rats tested (C and D). Amyloid plaques, as measured by staining the anti-Aβ antibody 6E10, were absent in all rats tested (E and F). Overall, histological analysis of these rats shows no evidence of neurodevelopmental impairments or FAD-like pathology at 15 days.To determine whether the biochemical changes caused by the analyzed A. Qualit3 region B. No evis tested , C and Ds tested , E and FPsen1 L435F mutation was selected, given its profound alteration of APP metabolism  PS1-L435F can assemble in a γ-secretase complex  It i pathway . Rat PseLF/LFPsen1 mice, both species were detected in this study at about 9% and 40% of WT controls for Aβ40 and Aβ42, respectively. In general, cell culture and in vitro models show that PS1 L435F mediates loss of Aβ40 and Aβ42 production. PS1 L435F–reconstituted PS1/2 KO mouse embryonic fibroblasts  is located on rat chromosome 6. We created a Long-Evans rat model with point mutation CTT→TTT at the rat Psen1 locus by CRISPR/Cas-mediated genome engineering. This mutation creates a rat that carries a Psen1 gene coding for PS1 with the FAD L435F mutation. The detailed procedures are reported in the Supporting Experimental Procedures.The rat m sucrose, 20 mm Tris-base (pH 7.4), 1 mm EDTA, and 1 mm EGTA plus protease and phosphatase inhibitors (Thermo Scientific), with all steps carried out on ice or at 4 °C. Total lysate was solubilized with 0.1% SDS and 1% NP-40 for 30 min while rotating. Solubilized lysate was spun at 20,000 × g for 10 min, the supernatant was collected and analyzed by ELISA and Western blotting.Rats were anesthetized with isoflurane and perfused via intracardiac catheterization with ice-cold PBS. Brains were extracted and homogenized using a glass–Teflon homogenizer (100 mg tissue/1 ml buffer (w/v)) in 250 mm urea) to 1× and loaded on a 4%–12% BisTris polyacrylamide gel . Proteins were transferred onto nitrocellulose at 25 V for 7 min using the Trans-blot Turbo system (Bio-Rad) and visualized by red Ponceau staining. Membranes were blocked for 30 min in 5% milk  and washed extensively in PBS/Tween 20 (0.05%), and primary antibody was applied overnight at 4 °C at 1:1000 dilution in blocking solution . The following antibodies were used: Tyr-188 , 6E10 , Pen2 , Presenilin 2 , Nicastrin , N-cadherin , Presenilin 1 C terminus , and Presenilin 1 N terminus . Anti-mouse  or a 1:1 mixture of anti-rabbit  and anti-rabbit , was diluted 1:1000 in 5% milk and used against mouse and rabbit primary antibodies for 30 min at room temperature with shaking. Blots were developed with West Dura ECL reagent  and visualized on a ChemiDoc MP Imaging System (Bio-Rad). Signal intensity was quantified with Image Lab software (Bio-Rad). Data were analyzed using Prism software and are represented as mean ± S.D.Biochemical analysis of rat brain samples was performed as described previously . BrieflyAppδ7/δ7 hypomorph control . Data were analyzed using Prism software and are represented as mean ± S.D.For analysis of Aβ38, Aβ40, Aβ42, sAPPα, and sAPPβ, the following Meso Scale Discovery kits were used. Aβ38, Aβ40, and Aβ42 were measured with V-PLEX Plus Aβ Peptide Panel 1 6E10 (K15200G) and V-PLEX Plus Aβ Peptide Panel 1. sAPPα and sAPPβ were measured with sAPPα/sAPPβ K15120E). Measurements were performed according to the manufacturer's recommendations. Plates were read on a Meso QuickPlex SQ 120. For analysis of Aβ43, the IBL Human Amyloidβ (1–43) (FL) Assay Kit (27710) was used according to the manufacturer's recommendations. The specificity of this kit was validated by us using a rat 0E. Measu control  ) with 1% CHAPSO, solubilized for 30 min at 4 °C while rotating, and spun at 20,000 × g for 10 min. Solubilized lysate was used as input for immunoprecipitation with anti-GFP , anti-PS1-CT, or anti-PS2-CT antibodies and protein A/G beads  overnight at 4 °C while rotating. After several wash steps, bound protein was eluted by 5-min incubation with 1× LDS sample buffer at 55 °C. Input and eluates were analyzed by Western blot analysis.Total brain lysate was diluted in immunoprecipitation buffer , and the appropriate TaqMan (Thermo) probes were used in the real-time PCR. Samples were analyzed on a QuantStudio 6 Flex Real-Time PCR System (Thermo), and relative RNA amounts were quantified using LinRegPCR software. The probe Rn00570673_m1  was used to detect rat Psen1, and samples were normalized to Gapdh levels, as detected with Rn01775763_g1 (exon junctions 2–3 and 7–8). Levels of Notch target gene transcripts were determined using the RT2 ProfilerTM PCR Array Rat Notch Signaling Pathway plate  according to the manufacturer's recommendations. Student's t test was used for all analyses, with data presented as mean ± S.D.Total brain RNA was extracted with the RNeasy RNA Isolation kit (Qiagen) and used to generate cDNA with a High-Capacity cDNA Reverse Transcription Kit (Thermo). 50 ng of cDNA, TaqManRat brain tissue was prepared and stained as described previously. Briefly, intracardiac paraformaldehyde-perfused rat brains were extracted and stored in 70% ethanol prior to cerebral coronal sectioning. Sections were dehydrated and paraffin-embedded and then processed into 15 cross-sections targeting the frontal cortex at the level of the isthmus of the corpus callosum and anterior and posterior hippocampus. IHC staining was performed in accordance with Biospective Standard Operating Procedure BSP-L-06. Slides were manually deparaffinized and rehydrated prior to automated IHC. Slides initially underwent antigen retrieval, either heat-induced epitope retrieval or formic acid treatment. All IHC studies were performed at room temperature on a Lab Vision Autostainer using the REVEAL Polyvalent HRP-AEC Detection System (Spring Bioscience). Briefly, slides were incubated sequentially with hydrogen peroxide for 5 min to quench endogenous peroxidase, followed by 5 min in Protein Block, and then incubated with primary antibodies . Antibody binding was amplified using Complement reagent (20 min) followed by an HRP conjugate (20 min) and visualized using the AEC Chromogen (20 min). All IHC sections were counterstained with Acid Blue 129 and mounted with aqueous mounting medium . The images underwent a quality control review, and final images were transferred to the Biospective server for qualitative image analysis. All qualitative assessments were performed blinded to the tissue genotype.i.e. when the ordinary one-way ANOVA showed statistical significance). Statistical analysis was performed with GraphPad Prism v8 for Mac. Significant differences were accepted at p < 0.05.Statistical significance was evaluated using ordinary one-way ANOVA followed by post hoc Tukey's multiple comparisons test when applicable (All of the data are contained in the manuscript.M. D. T. and L. D. conceptualization; M. D. T. and L. D. data curation; M. D. T. and L. D. formal analysis; M. D. T. and L. D. validation; M. D. T. and L. D. investigation; M. D. T. and L. D. visualization; M. D. T. and L. D. methodology; M. D. T. and L. D. writing-original draft; M. D. T. and L. D. writing-review and editing; L. D. resources; L. D. supervision; L. D. funding acquisition.