Programmed cell death 1 ligand 1 (PD-L1) is a ligand of programmed cell death 1 (PD-1) that functions as an immune checkpoint by down-regulating immune responses. To determine whether PD-L1 is a therapy target in vitiligo treatment, Pmel-1 vitiligo mice were treated with a PD-L1 fusion protein. Treatment with this fusion protein significantly reversed/suppressed depigmentation development in adult Pmel-1 mice. Mechanistically, enrichment of regulatory T cells (Treg) in the skin was detected after PD-L1 fusion protein treatment in Pmel-1 mice. Furthermore, Tregs abundance was also increased in both the spleen and circulation of Pmel-1 mice treated with PD-L1. These data indicate that PD-L1 protein therapy inhibits the immune response and reverses depigmentation development in Pmel-1 vitiligo mice. The etiology of vitiligo remains unclear. However, the critical role of melanocyte-reactive T cells is well recognized. Histological and genetic studies have demonstrated that vitiligo skin is populated with inflammatory lymphocytic infiltrates consisting of both activated CD4+ and CD8+ T cells2. Multiple factors, including genetic factors and environmental stimulation, activate the T cell-specific killing of epidermal melanocytes (MC) that ultimately leads to their death3. Different Treg activation strategies, such as CCL22 up-regulation4 and simvastatin administration3, have demonstrated significant treatment effects in reversing depigmentation in h3TA2 and Pmel-1 vitiligo mice. However, despite the current availability of drug treatments, physical therapy, surgical treatments and other methods, clinical efficacy is not satisfactory. New therapeutic strategies should be evaluated and tested for patients with vitiligo.Vitiligo is a common, disfiguring autoimmune disease of the skin and is characterized by gradual skin depigmentation5. In response to binding by its ligands, programmed cell death 1 ligand 1(PD-L1) (B7-H1 or CD274) and PD-L2 (B7-DC or CD273)7, PD-1 represses the activation and function of T cells5. PD-1 is not usually expressed on resting T cells, but its expression is induced after T cell activation, for example, by ligation of the T cell receptor (TCR)9. The absence of PD-1 rendered mice resistant to viral infection and suppressed tumor growth and tumor metastasis by reducing the antigen-recognition threshold and increasing the cytotoxic activity of CD8+ T cells. PD-1/PD-L1 pathway-targeted therapies are under development in multiple diseases, including different cancers. Keytruda  is a humanized monoclonal IgG4 PD-1 antibody and has been approved by the FDA since September 4, 2014 for patients with advanced or unresected melanoma that no longer responds to other drugs.Programmed cell death 1 (PD-1) is a member of the extended CTLA-4 family and is a receptor that, functioning as an immune checkpoint, down-regulates immune responses10. The PD-L1-targeted therapy has also been demonstrated to be effective in experimental psoriasis, a chronic inflammatory skin disorder11. Overexpression of PD-1 protein was detected in the skin of the Imiquimod (IMQ)-induced murine model of psoriasis12, in which inflammation was inhibited by injecting a recombinant PD-L1 protein11. Further experiments indicated that severe psoriatic inflammation was induced in PD-1-null mice12. Interestingly, the PD-L1 fusion protein is effective in inhibiting T cell activity in skin autoimmunity disease. Thus, we have a strong rationale to test whether the PD-L1 fusion protein could be a new therapeutic strategy in patients with vitiligo.PD-L1, also known as B7-H1 and CD274, is a transmembrane glycoprotein and a member of the B7 family of immune regulatory molecules with an approximately 65 kDa. Mature human PD-L1 consists of a 220 amino acid (aa) extracellular domain (ECD) with two immunoglobulin-like domains, a 21 aa transmembrane segment, and a 31 aa cytoplasmic domain, in which shares 73% and 74% aa sequence identity with mouse and rat PD-L1, respectively. B7-H1 is expressed on inflammatory-activated immune cells including macrophages, T cells, and B cells, keratinocytes, enothelial and intestinal epithelial cells, as well as a variety of carcinomas and melanomaHere, we tested the treatment effect of PD-L1 fusion protein in Pmel-1 vitiligo mice. We found that PD-L1 fusion protein treatment reversed depigmentation via a markedly increased abundance of Tregs in the skin.in vivo using Pmel-1 mice, representing a slowly depigmenting model. PD-L1-Fc was administered intraperitoneally at 20 µg/injection 3 times a week for 6 weeks. The recombinant mouse PD-L1/Fc used here is a disulfide-linked homodimeric protein after proteolytic removal of the signal peptide. To produce these recombinant protein, a DNA sequence encoding the extracellular domain (Met 1-Thr 238) of mouse PD-L1 (NP_068693.1) was fused with the C-terminal His-tagged Fc region of human IgG1 at the C-terminus. The PD-L1-Fc has strong affinity with its receptor, PD-1. A linear range of 3.2–400 ng/ml PD-L1-Fc can binds with immobilized recombinant mouse PD-1 at 10 μg/ml (100 μl/well) . Depigmentation was observed and quantified once a week for all periods of treatment and for 10 weeks after the last PD-L1 fusion protein injection. Compared with control IgG treatment, PD-L1-Fc treatment significantly reversed depigmentation after the last injection, as represented in Fig. First, we investigated the therapeutic effect of PD-L1-Fc 14. Thus, Tregs are major foci in the study of autoimmune pathogenesis of vitiligo15. We first detected the expression of PD-L1 protein in isolated epidermal melanocytes in Pmel-1 mice with specific anti-PD-L1 antibodies using flow cytometry. We found that the expression of PD-L1 protein was repressed in Pmel-1 Mouse Primary Melanocytes (MPM) vs. WT MPM , characterized by expression of the forkhead box P3 (FoxP3) signature transcription factor, are crucial for maintaining peripheral tolerance and restraining autoreactive T cells from mediating autoimmune responsesMPM Fig. .Figure 2+CD3+ Treg cell populations were increased in splenocytes (*P < 0.05) by PD-L1 stimulation. Specifically, average Treg percentages among total T cells in the spleens were 2.7 +/− 1.3% and 11.4 +/− 2.2% in the vehicle group (n = 6) and the PD-L1 group (n = 6), respectively  compared to the IgG control group (35.1 +/− 7.9 cells/mm2)  . The numbers of TCR-positive cells were quantified. We found that the abundance of Vβ12-expressing T cells was reduced by 61.4% in the skin of PD-L1 fusion protein-treated mice (13.4 +/− 5.1 cells/mm16) Fig. . These rp = 0.003) . Cells were then incubated with cognate peptide prior to quantification of IFN-γ-secreting T cells by ELISPOT. As shown in Fig. 03) Fig. . These r16. Useful vitiligo therapeutic strategies should inhibit disease progression and promote repigmentation through simultaneous melanocyte regeneration, proliferation and migration. The center of the effective treatment strategy is to repress melanocyte-reactive T cells18. Here, we found that PD-L1 fusion protein treatment repressed the abundance of melanocyte-reactive T cells in vitiligo in vivo, providing a new potential therapeutic strategy for patients with vitiligo.The pathogenesis of vitiligo involves interactions between intrinsic and extrinsic melanocyte defects, innate immune inflammation and T cell-mediated melanocyte destruction20, another skin autoimmune disease with T cell activation. Furthermore, several independent groups have demonstrated that PD-L1 fusion protein treatment is effective in experimental psoriasis in vivo21. In addition, PD-1 antibody treatment resulted in vitiligo development in clinical trials of cancer22.The effect of PD-L1 fusion protein treatment on vitiligo was expected. PD-L1 is a ligand of the PD-1 receptor, and PD-1 is a member of the extended CD28/CTLA-4 family of T cell regulators. Both CTLA-4 and PD-1 are receptors that negatively regulate T cell activation. Interestingly, soluble chimeric protein CTLA4Ig (BMS-188667) treatment achieved a 50% or greater sustained improvement in clinical disease activity, with progressively greater effects observed in the highest dosing cohorts in patients with psoriasis14. A critical role of Tregs in vitiligo has been demonstrated by histological studies in humans and in h3TA2 (human TIL derived Tyrosinase TCR transgenic on HLA-A2) vitiligo mouse models15. Here, we further demonstrated that Treg repression is also a key factor in Pmel-1 vitiligo development, in which some spontaneous depigmentation of the pelage is observed beyond 20 weeks, and vitiligo develops slowly. Activation of Treg has been recognized as an effective treatment strategy in vitiligo treatment. As a targeted treatment strategy, CCL22 DNA has been demonstrated to promote Treg skin homing to suppress depigmentation4. Furthermore, simvastatin treatment also represses effector T cells and activates Treg to reverse depigmentation3. Here, we found another targeted treatment strategy in vitiligo treatment, as PD-L1 fusion protein treatment had a similar treatment effect as CCL22 and simvastatin. However, effective treatment with the PD-L1 fusion protein was more stable than either of them. Specifically, we found that the depigmentation suppression effect was observed until 8 weeks after the final treatment with PD-L1 fusion protein compared with <2 weeks and 1 only week for CCL22 DNA and simvastatin treatments, respectively. However, none of them yielded a permanent repigmentation effect. Thus, combination therapies will be required to address all aspects of vitiligo pathogenesis to obtain the greatest treatment efficacy.Regulatory T cells (Treg) are characterized by expression of the FoxP3 signature transcription factor, high levels of surface CD25 for IL-2 uptake and other surface molecules and play a pivotal role in maintaining peripheral tolerance and restraining autoreactive T cells from mediating autoimmune responses6 cells/well and cultured in RPMI 1640 with 10% FBS and antibiotics . IFN-γ production was measured directly from cultured splenocytes of treated mice. ELISPOT kits were purchased from MABTECH . Briefly, plates were coated with captured anti-IFN-γ antibodies. IFN-γ-secreting cells were detected using biotinylated anti-cytokine antibodies, followed by horseradish peroxidase-conjugated streptavidin (MABTECH) and 3-amino-9-ethylcarbazole substrate .Splenocytes isolated from Pmel-1 mice  were seeded at 1 × 1023 were obtained from the Jackson Laboratory . In an intervention experiment using neutralizing mAb against PD-1, an mPD-L1 fusion protein (50010-M03H-200) was purchased from Sino Biological Inc. Mice were i.p. injected with 200 µg PD-L1 fusion protein or IgG2a on days 1, 3 and 5 of each week for 6 weeks. Mouse fur color and weight were observed once a week. All mouse procedures were approved by the Yueyang Hospital IACUC. All methods were performed in accordance with the relevant guidelines and regulations of the Yueyang Hospital IACUC.Pmel-1 mice with a gp100-reactive transgenic T cell receptorMouse dorsal skins were formalin-fixed and paraffin-embedded. Hematoxylin and eosin (H&E) staining of paraffin-embedded section (4–5 μm) was conducted according to standard methods. Immunohistochemical staining was performed following the regular procedure with the following antibodies: anti-CD3 (sc-20047), anti-CD4 (sc-7219) and anti-CD8 (sc-7188). Isotype controls (Santa Cruz) were included in each staining. Slides were examined using an Olympus microscope. Quantification of H&E and IHC staining was performed by two experimenters for two sections of two mice per treatment group. Numbers of positive cells were counted for five high power fields (HPFs) and averaged. Immunofluorescent staining was performed following the regular procedure with the following antibodies: anti-CD3 (145-2C11), anti-CD4 (RM4-5), and anti-CD25 (PC61) antibodies .Cells were stained in flow cytometry staining buffer  for 30 minutes. The antibodies used included IFN-γ-PE-Cy7 . All cells were blocked with Purified Human Fc Receptor Binding Inhibitor .p < 0.001, p < 0.01 and p < 0.05 were marked by three, two and one asterisk, respectively.The statistical significance of data was determined using a two-tailed unpaired Student’s t-test. The correlation between positive cell numbers of different markers was evaluated by Pearson’s correlation coefficients. Values of


Recently, some studies have demonstrated an improved performance using L1-regularization  related to an increase in the spatial resolution and sensitivity of the recovered traction field. In this manuscript, we present a comparison between the previous two regularization schemes (relying in the L2-norm for the data fidelity term) and the full L1-regularization (using the L1-norm for both terms in the cost function) for synthetic and real data.Traction Force Microscopy (TFM) is a widespread technique to estimate the tractions that cells exert on the surrounding substrate. To recover the tractions, it is necessary to solve an inverse problem, which is ill-posed and needs regularization to make the solution stable. The typical regularization scheme is given by the minimization of a cost functional, which is divided in two terms: the error present in the data or data fidelity term; and the regularization or penalty term. The classical approach is to use zero-order Tikhonov or L1-regularizations give an improved spatial resolution (more important for full L1-regularization) and a reduction in the background noise with respect to the classical zero-order Tikhonov regularization. In addition, we present an approximation, which makes feasible the recovery of cellular tractions over whole cells on typical full-size microscope images when working in the spatial domain.Our results reveal that L1-regularization improves the sensitivity to recover small stress footprints. Moreover, the proposed method has been validated to work on full-field microscopy images of real cells, what certainly demonstrates it is a promising tool for biological applications.The proposed full LThe online version of this article (doi:10.1186/s12859-017-1771-0) contains supplementary material, which is available to authorized users. Tissue remodeling implies the reorganization of the extracellular matrix (ECM), which is driven by the conversion of intracellular-generated mechanical forces into extracellular traction, which reorganizes the ECM fibers. This is a crucial process during regeneration  but it is equally important in pathologic scenarios . In fact, some of the pathological changes associated to these diseases are due to the altered ability of cancer, or inflammatory cells, to exert abnormal traction on their microenvironment . Thus, tTraction Force Microscopy (TFM) is a technique that estimates the tractions exerted by adherent cells placed on top of a flexible hydrogel that mimics the mechanical properties of the ECM .In a typical TFM experiment, a large number of fluorescent beads are randomly mixed inside the hydrogel. The fluorescent beads are displaced as the cells exert tractions on the substratum and act as fiduciary markers enabling the transformation of motion into force see Fig. . ExperimThe displacement field of the beads can be obtained using different image processing methods. Two classical approaches used in TFM experiments include block-wise image correlation computation (Particle Image Velocimetry) ; and indRecovering cellular tractions (causes) from matrix displacements (observable effects) implies solving an ill-posed inverse problem. Consequently, the solution is very sensitive to the errors committed in the calculation of the displacement field.2-norm regularization, also known as Tikhonov regularization  is a 2N × 1 column vector with N being the number of locations where displacements are measured, M × 1 column vector with M being the number of locations where tractions will be estimated, and K is the 2N × 2M stiffness matrix given by:In practice, displacement and traction fields are only estimated at discrete locations of the space, and Eq.  can be rq denotes the Lq-norm, ∥Kt − u∥q is the data fidelity term, ∥t∥p is the imposed penalty and λ is a parameter that controls the amount of regularization applied.A trivial solution for the traction recovery is obtained by the direct inversion of Eq. 2-regularization has an efficient, and widely used, implementation in Fourier domain  with a step size Δλ of 0.01. The regularization parameter λ used on the simulations for the evaluation of the algorithms was the one minimizing the cost functional defined on Eq. We have evaluated the error in the recovered traction field within a region of interest defined by the corresponding stress footprint. These footprints were obtained by segmenting the magnitude of the recovered tractions using an Isodata thresholding of two levels. These metrics are evaluated only in the recovered stress footprint areas.tgt the simulated ground-truth traction field, Being Pn the points of the simulated stress footprint associated to each recovered footprint.Absolute error in the recovered traction magnitude (in percentage) defined asAverage angular error (in percentage) in the recovered stress footprint, weighted at each point by the traction magnitude. It is normalized by the maximum error in degrees (180°) to give the result in percentage as given byAgt being the area of the recovered and ground-truth footprints, respectively.Absolute error of the recovered stress footprint area (in percentage) as given byngt are the number of recovered and ground-truth stress footprints.It is the ratio (in percentage) between stress footprints that the regularized TFM methods cannot recover  and the total number of simulated stress footprints synthetized by cell. It is defined asμm2) and the peak magnitude (in kPa) of the recovered stress footprint areas. This metric reflects the dependence between the size and the magnitude of the stress footprints and it is defined asIt is the smallest product between the area . Images of the cells were acquired at multiple locations of the hydrogel surface with a 60× glycerol objective (NA = 1.3) mounted in a Leica SP5 Laser Scanning Confocal microscope with 0.32 μm of resolution in each direction of the plane. Time-lapse images of the stressed hydrogel were taken every 20 s during 25 min. An image of the relaxed hydrogel was taken after removing the cells.We have used the presented algorithms to quantify the tractions exerted by Chinese Hamster Ovary , CCL 61) cells expressing Lifeact-GFP. These cells were cultured on a ~ 90 μm thick polyacrylamide hydrogel with a Young’s modulus of 5 kPa and a Poisson ratio of 0.45, containing embedded fluorescent polystyrene beads  realizations were simulated for each synthetic case to take into account the variability introduced by the random distribution of the beads inside the hydrogels. Finally, for the sake of comparison, the parameters controlling the amount of regularization were fixed for all the set-ups to To build a realistic synthetic dataset we used our TFM simulator with data from ten different real TFM experiments  as explained in the previous section. A wide variety of test conditions were considered, specifically, the number of stress footprints per simulated case ranged between 10 and 120, their peak magnitude between 0.6 kPa and 4 kPa and their area between 0.25 μm1-regularizations give sparser traction fields with less background signal and better estimation of traction magnitudes than the classical L2-norm based Tikhonov regularization. Furthermore, full L1-regularization is able to recover more stress footprints than the other methods.Figure 1-regularization presents smaller errors in magnitude and angle of the recovered stress footprint areas than Tikhonov regularization and full L1-regularization, giving around three times smaller magnitude error (12%) than the other methods. Regarding the area error of the recovered stress footprints, full L1-regularization shows the best performance with an average error less than 4%, while L1-regularization has about 7% of error and Tikhonov present the worst performance with an average area error about 16%. Looking at the ratio of the lost stress footprints, full L1-regularization is able to recover more than the double of stress footprint areas (around 75%) than the other regularization methods (around 30% of them). In addition, in Fig. 1-regularization has the smallest detectable stress footprint area values. Namely, this method detects smaller stress footprint areas with reduced traction magnitude than the other methods. In particular, the smallest stress footprint areas detected in all experiments for each method are (Peak Magnitude (kPa), Area (μm2), mFA(nN)): L2-regularization ; L1-regularization ; full L1-regularization .Those results are quantitatively confirmed in Fig. p < 0.001). The exceptions are: The error in magnitude between Tikhonov and full L1-regularization and the smallest footprint detected between Tikhonov and L1-regularization.Moreover, most of the differences between the error measures given for the different regularization schemes are statistically significant  as well as outliers. In these cases, applying the L1-norm data fidelity constraint is more appropriate.The use of the full Ltroduced  and has troduced –28, imagtroduced , 30 and troduced . Whereas1 norm in the data fidelity for image reconstruction [1 fidelity based model over the standard one is that the regularization imposed on solutions is more geometric, in the sense that the regularization process has less dependence on the contrast of image features than on their shapes; As distinct from the standard model, small features in the images maintain their contrast. In addition, L1 data fidelity works better reducing some artifacts like ringing and the presence of outliers [Moreover, there has been previous theoretical work exploring the consequences of the use of the Ltruction . In partoutliers , 29.1-regularization reduces the data fidelity term. This means that the matching between the estimated displacements and the inferred ones  is greatly improved with respect to the other regularization schemes. This could be responsible for the strong decrease in the loss stress footprints ratio and it is the source of differences between the L1-regularizations.In Additional file 1-regularizations give an improved spatial resolution (more important for full L1-regularization) in terms of sensitivity to detect smaller stress footprints in both synthetic and real data with respect to the classical zero-order Tikhonov regularization.Our results reveal that L1-regularization of the penalty term presents smaller errors in magnitude and angle of the recovered stress footprint areas than the other regularization methods. It was expected that L1-regularization of the penalty term over-performed the results obtained by Tikhonov regularization as was already demonstrated in previous publications [1-regularization and it is maybe due to our error metric evaluation procedure. Namely, we compute the error metrics over all the recovered stress footprints being the number recovered by full L1-regularization much larger. Even more, the ones recovered only by full L1-regularization tend to be the smaller in magnitude and area. Those risk to be the ones over which the reconstruction errors could be larger penalizing the computation of the error metrics.In our hands, Lications , 18. Nev2-regularization in the Fourier domain  and in the spatial domain (using just the data covered by the mask) are visually similar, and no loss of information is apparent as shown in Fig. p < 0.001) when computed on our synthetic dataset. As this solution is largely conservative, we believe that further refinements could reduce the computational demand even further. Finally, there exists an alternative TFM strategy called Traction Reconstruction with Point Forces (TRPF) [As detailed in the previous sections, working in the spatial domain implies a high computational demand in terms of RAM and CPU usage due to the size of the stiffness matrix and the need to use an iterative optimization algorithm to recover the tractions, respectively. To overcome this severe problem, we have presented a method to reduce the size of the stiffness matrix that allows working with full-field microscopy images and full resolution displacement fields in the spatial domain. It is based on the calculation of forces in a few confined regions of interest defined with an Otsu thresholding over the magnitude of the estimated displacements. This approach results in a generous segmentation of the regions, guaranteeing no loss of information. Namely, more than 85% of the estimated displacements magnitudes are kept in all cases. This can be qualitatively observed in Fig. s (TRPF)  that alss (TRPF) .Fig. 8Co2-regularization in Fourier Domain ; L2-regularization in spatial domain ; L1-regularization in spatial domain ; full L1-regularization in spatial domain .Recent publications have implemented novel methods incorporating relevant biomechanical constraints  , 35. In 1-regularization methods , a more efficient implementation could be performed in a more suitable platform , which would take advantage of every available resource of a desktop computer.To reduce the computation time of the LIt is worth noting that we have simulated very small stress footprints in some of the synthetic traction maps. Those are very difficult to recover due to a variety of experimental constraints, namely, the random position of the beads, the limited microscope resolution and the possible proximity of other stress footprints with higher size or magnitude. A limitation of our experimental set-up is the fact that we use relatively large beads (200 nm) restraining the density of fiducial markers embedded in the hydrogel to avoid clustering. This issue could be partly solved by using smaller beads (~40 nm) that could be packed with a higher density and closely to the surface . A recenAs per the experiments with real data we have only performed a qualitative comparison of the different regularization methods. Our current work focuses on designing experiments in which the cellular focal adhesions will be stained in vivo. For this purpose, we are constructing cells stably expressing bona fide markers of focal adhesion coupled to fluorescent markers (e.g. EGFP), which are commonly used in these types of experiments . Our motWe believe this improvement in sensitivity and spatial resolution could be relevant when combined with advanced microscopy methods, for cell biomechanics studies willing to drive the focus toward the characterization of individual focal adhesions instead of clusters of them as historically performed.1-regularization to recover cellular tractions in TFM experiments, showing its good performance compared with classical Tikhonov regularization and simple L1-regularization both in simulated and real data.In this manuscript, we have implemented full L1-regularization is the huge improvement in the sensitivity and the spatial resolution, which allows recovering smaller stress footprints compared with the state-of-the-art regularization methods. In addition, we have demonstrated the improved performance of L1-regularizations, giving smaller errors in the recovered traction field and resulting in less background noise than Tikhonov regularization.The main characteristic of full LIn this work, we have also presented a method to make feasible the recovery of the tractions exerted by whole cells on full-field microscope images when working in the spatial domain. We have further demonstrated the suitability of the approach for the analysis of both realistic simulations and real data experiments.Additional file 1:λ) values. The first row is for Tikhonov regularization, the second one for L1-regularization and the third row is for full L1-regularization. The columns from left to right show: traction field (in Pa), displacement field (in μm), K ∙ t in Eq. Kt − u||q in Eq. Comparison of the different terms involved in the cost functional for the different regularization methods and a swept of the regularization parameter  as computed by a Student’s test. (TIFF 431 kb)Quantitative results from synthetic data. Table with the different error metrics (mean±standard deviation) obtained by the different regularization methods on the synthetic data. Ten different traction maps have been considered and ten realizations for each one of them. The best results for each metric are highlighted in red. For all cases, the differences between the realizations are statistically significant  full L1-norm regularization. The outline of the mask used for traction recovery is shown in red  and white (for the rest). The scale bar represents 30 μm. Frame #5 corresponds to Fig. Ten samples frames illustrating the whole TFM experiment and comparing the traction recovery with the different regularization methods. For each frame:  CHO cell expressing Lifeact-GFP;  Pseudo-color image showing the fluorescent beads at the hydrogel surface. The beads of the unstressed and stressed hydrogels have been superposed and pseudo-colored in red and green, respectively; therefore, beads are colored in yellow when not displaced. The contrast of the pseudo-color images has been modified to highlight the areas with bead displacements;  Magnitude (in μm) and direction (arrows) of in-plane displacements estimated from the bead images. (Bottom row) Recovered traction magnitude (in Pa) and direction (arrows) using: (Left) Tikhonov regularization; (Center) LAdditional file 4:Force balance over real cells. Table with the mean (in Pa and in percentage of the maximum traction magnitude) and the standard deviation (in Pa) of the sum of forces over the whole cell for each regularization scheme and for all real dataset. (TIFF 110 kb)


In addition, using a trilayer graphene, mono- and bi-layer graphene could be successfully fabricated after one- and two-cycle ALE of the trilayer graphene, respectively. We believe that the ALE technique presented herein can be applicable to all layered materials such as graphene, black phosphorous and transition metal dichalcogenides which are important for next generation electronic devices.The electronic and optical properties of graphene are greatly dependent on the the number of layers. For the precise control of the graphene layers, atomic layer etching (ALE), a cyclic etching method achieved through chemical adsorption and physical desorption, can be the most powerful technique due to barely no damage and no contamination. In this study, we demonstrated the ALE process of graphene layers without noticeably damaging the graphene by using a controlled low energy oxygen (O Especially, its electronic and optical properties greatly depend on the number of layers in graphene9. Accordingly, the accurate control of the number of layers is the most important technology in various of graphene-based device applications.Graphene has attracted considerable attention due to their unique properties including very high electron mobility, extremely high mechanical strength, superior thermal conductivity, and high chemical stability10, Joule heating11, laser thinning13, plasma etching using helum, nitrogen and oxygen18. However, these techniques can be difficult to achieve precise control of graphene layers at the atomic layer scale because the removal rate of graphene layers depends only on etch time. Also, for some methods, it is even difficult to remove the graphene layers without inducing damage and contamination to the surface.To control the layers of graphene, researchers have investigated various methods such as oxidation etching in a tube furnaceet al.19 by the deposition of Zn on a photoresist patterned graphene surface, lift-off Zn on photoresist, and by the removal of patterned Zn on graphene surface with one graphene layer using HCl. Even though this method can remove graphene layers layer-by-layer precisely without damaging the graphene layer surface, it may not be applicable for nanoscale electronic device fabrication due to the requirement of a lift-off process used for patterning Zn on the graphene surface. Atomic layer etching (ALE), a cyclic etch method composed of chemical adsorption and physical desorption of the sequential steps, is also one of the techniques that have good potential in precisely removing graphene layer-by-layer without inducing damage and contamination. In addition, it is applicable for the nanoscale device fabrication because it removes graphene layers after photoresist patterning using coventional patterning methods.Precise control of graphene layers by layer-by-layer removal has been reported by Dimiev 2+/O+)-ion for chemical adsorption and a low energy Ar+-ion beam for physical desorption, we were able to achieve a precise control of graphene layers without damage on the bottom graphene layer. Especially, for the photoresist patterned graphene or the chemical vapor deposited (CVD) graphene transferred from the Cu using Poly(methyl methacrylate)(PMMA), a thin polymer residue is remaining on the graphene surface even after the removal of the photoresist/PMMA. Using the low energy Ar+-ion, the polymer residue layer could be also completely removed in this study before the ALE process without damaging the graphene surface21.In this research, layer-by-layer removal of graphene using ALE process using an ion beam has been investigated for the various next generation nanoscale graphene electronic devices. Using an optimized ALE process composed of a controllable low energy oxygen, and immersed into a FeClThe schematic diagram of a two-grid (4inch-diameter) inductively coupled plasma (ICP)-type ion beam system with axial magnetic field used for the layer-by-layer removal of graphene, that is, for graphene ALE and a quadrupole mass spectrometer (QMS) installed for the measuremet of ion energy and flux of the ion beam is shown in Fig. 2+/O+)-ion and a low energy Ar+-ion beam, the 1st grid of the ion beam source was floated without applying any voltage while the 2nd grid was grounded. The details of the ion beam system can be found elsewhere21. In some cases, an axial magnetic field of 30 Gauss was applied in front of the 1st grid to decrease the ion beam energy. To measure the ion energy distribution on the graphene surface distribution at various ion beam conditions such as different radio frequency (rf) powers and gas flow rates to the ion source and with/without axial magnetic field, a mass spectrometer was installed at the substrate location. As a result of this analysis, the following processes were used as the optimized ALE cyclic steps; for chemical adsorption step, an oxygen-ion beam generated with 100 sccm of O2, 15 W of rf power, and with axial magnetic field of 30 Gauss for 90 s and, for the physical desorption step, an Ar+-ion beam generated with 100 sccm of Ar and 500 W of rf power for 120 s.To obtain a low energy oxygen calculation with a local-density approximations (LDA)23 and the projector-augmented wave (PAW)24 method. A 7 × 7 × 1 grid for k-point25 sampling and the energy cut-off of 400 eV were consistently used in our calculations. The convergence threshold for energy was set to 10−5 eV.To investigate the graphene ALE mechanism by the chemical adsorption of oxygen and physical desorption by Ar ion bombardment, a computational simulation on the change of binding energies between carbon atoms on graphene before and after the oxygen adsorption was performed using Vienna Ab Initio Simulation Package (VASP)The lattice constant of graphene unit cell was calculated to be 2.45 Å after relaxation. Then, geometry optimization was carried out on the 3 × 3 × 1 supercell structure. To describe the bilayer graphene, two-dimensional periodic boundary conditions were used along the growth direction with vacuum space of at least 10 Å to avoid additional interaction between the layers. For the bilayer graphene structure, both A-A stacking and A-B stacking structure were considered  was calculated by EB = (Etot − Esub − ES − EvdW)/n, where Etot, Esub, ES, and EvdW stand for the electronic energy of the entire system, the system after removing the target atom, the target atom, van der Waals interaction, respectively, and n stands for the number of atoms bonded to the target atom. Here, the van der Waals interaction energy between the target atom and adjacent layer was calculated by EvdW = (ES+AL − EAL − ES), where, ES+AL and EAL stand for the electronic energy of the system (adjacent layer and the target atom are held together) and the energy of the adjacent layer, respectively.The binding energy of an atom (E+) at a wavelength of 532 nm and atomic force microscopy . Optical transmittance of the graphene layers was measured by Ultraviolet-visible-near infrared (UV-Vis-NIR) absorption spectroscopy. For optical trasmittance, the trilayer graphene was deposited on glass substrates.Graphene surface was examined with Raman spectroscopy  and Ar+-ions extracted from the ion source for the operation of sequential steps of ALE composed of chemical adsorption by O2+-ion and physical desorption by Ar+-ion. The 1st and 2 nd grid voltages of the ion gun were floated and grouned, respectively, for low ion energy operation and the rf power or gas flow rates were varied. Figure 2+-ion energy distribution measured for different rf powers at 100 sccm of O2 gas flow rate, the Ar+-ion energy distribution for different rf powers at 70 sccm of Ar gas flow rate, and the Ar+-ion energy distribution for different Ar flow rates at 500 W of rf power, respectively. As shown in Fig. 2+-ion energy was spread over a wide range with dual peaks and the decrease of rf power from 100 to 15 W decreased the ion energy. When an axial magnetic field of 30 G was applied to the ion source during the operation at 15 W, the energy was further decreased while having the energy distribution in the range from 0 to 20 eV. Figure 2+-ions in Fig. 2+-ion energy distribution showing dual ion energy peaks, the increase of ion energy and flux with the rf power suggest the characteristics of capactively coupled plasma (CCP) for oxygen plasma not ICP possibly due to low electron density for oxygen plasmas. To use a low energy oxygen-ion for chemical adsorption step, we used the condition of 15 W of rf power with 30 G of magnetic field at 100 sccm of O2 gas flow rate. In the case of Ar+-ion, as shown in Fig. +-ion, the increase of rf power decreased ion flux to the substrate. The differences between Fig. +-ion energy may be required but, for the physical desorption of chemisorbed species, a sufficient Ar+-ion energy is required. Therefore, to investigate the optimized Ar energy condition for the phyiscal desorption of graphene ALE, we etched the graphene surface with the condition in Fig. +-ion energy in the range from 11.2 to 14.1 eV.Figure 2 gas flow rate. For comparison, the Raman spectrum of a pristine monolayer graphene was included. The Raman spectra were normalized by G peak at 1580 cm−1 to compare the intensity of D peak and 2D peak accurately. As shown in Fig. −1 and decreased the 2D peak at about 2675 cm−1, however, the further increase of adsorption time to 120 s did not change the peak intensities. The increase of D peak and the decrease of 2D peak with the increase of oxygen-ion adsorption time is believed to related to the oxygen chemisorption on the monolayer graphene network and no further change of D and 2D peak intensities after 90 s suggests the saturation of oxygen chemisorption on the monolayer graphene surface. No change of 2D peak intensity after 90 s also reveals the existence of stable graphene structure up to exposure to 120 s27. Figure +-ion energies from 11.2 to 14.1 eV in Fig. +-ion energy as shown in Fig. +-ion, very small increase or no significant change  of D peak was observed while the increase of Ar+-ion energy signficantly increased the D peak intensity while decreasing the 2D peak intensity indicating significant damage to the graphene surface with increasing the Ar+-ion energy. As the results of Fig. 2+-ion was optimized at 90 s and the Ar+-ion desorption energy at 11.2 eV.Figure 2+/O+)-ion  and the physical desorption using Ar+-ion (500 W and 100 sccm of Ar for 120 s) and the results are shown in Fig. +-ion beam, one monolayer graphene could be completely removed indicating successful ALE process for graphene.A pristine monolayer graphene was sequentially exposed using the optimized conditions of the chemical adsorption using oxygen , and 150 s (overetch condition). As shown in Fig. +-ion exposure time to 120 s, the D peak intensity was decreased significantly near to zero due to the removal of the top chemically modified graphene layer on the bilayer graphene while increasing the 2D peak intensity due to the formation of monolayer graphene from bilayer graphene. The increase of Ar+-ion desorption time further to 150 s after the removal of one monolayer graphene on bilayer graphene did not change the Raman peak intensities indicating no damage or change of graphene structure exposed after the ALE.Using a pristine bilayer graphene, one cycle of graphene ALE was processed and the Raman data of the processed bilayer graphene during each step of one ALE cycle are shown in Fig. +-ion exposure time from 60 to 150 s, and the results are shown in Fig. −1, however, after the 90 s exposure to the oxygen-ion during the chemisorption step, the peak position was red shifted to 2675 cm−1. The exposure to the Ar+-ion for the desorption of C-O chemisorbed species to 90 s further red shifted the 2D peak to 2668 and 2657 cm−1 for 60 s and 90 s, respectively. The red shift of 2D peak after the exposure to the oxygen-ion and Ar+-ion is believed to be related to the strain of C-C bonding in the low binding energy C-C structure of the top graphene layer by the C-O bonding and Ar ion bombardment29. However, as the top graphene layer is removed and as the second graphene layer is exposed by the Ar+-ion exposure for 120 s, the 2D peak position was recovered similar to that of the pristine bilayer graphene. The 2D peak position after Ar+-ion exposure of 120 s was about 4 cm−1 lower than that of pristine bilayer graphene because the graphene layer changed from a bilayer to monolayer as seen in Fig. 30. When the Ar+-ion exposure time was further increased to 150 s, no change of 2D peak position was also observed. In addition, as the graphene layer is decreased from bilayer to monolayer by the Ar+-ion exposure, the full width at half maximum (FWHM) of the G and 2D peaks was decreased after the formation of monolayer graphene after the Ar+-ion exposure of 120 s and the further increase of the exposure time to 150 s did not change the FWHM, either. The FWHM of the G and 2D peaks of pristine bilayer graphene after one cycle ALE decreased from 22 to 18 cm−1 and from 48 to 28 cm−1, respectively  was decreased to 0.437, 0.285, and 0.130, respectively. Especially, the I(D/G) for the Ar+-ion exposure of 120 s was similar to that of the pristine graphene indicating the removal of chemically modified top graphene layer. Even after Ar+-ion exposure for 150 s, I(D/G) was 0.131, indicating no change in D peak  by the overexposure by Ar+-ion possibly due to a low energy Ar+-ion beam (11.2 eV). In the case of I(2D/G) in Fig. +-ion for 60, 90, and 120 s, the I(2D/G) was increased to 1.310, 1.515, and 1.773 showing the formation of monolayer graphene by the removal of top graphene layer27. In addition, I(2D/G) after Ar+-ion desorption further to 150 s did not change the I(2D/G) which shows that the ALE process  was completed at Ar+-ion exposure time of 120 s.Figure 2, 15 W of rf power, and with axial magnetic field of 30 Gauss for 90 s and, for the physical desorption step, an Ar+-ion beam generated with 100 sccm of Ar and 500 W of rf power for 120 s) could also confirmed by etching trilayer graphene and, by observing the change of the optical transmittance at 550 nm. After one, two, and three cycles of ALE using trilayer graphene, the optical transmittance of the etched graphene at 550 nm increased to 94.7, 97.2, and 99.7%, respectively, indicating the formation of bilayer, monolayer, and no layer, therefore, one monolayer removal for one cycle ALE could be confirmed -ion (0~20 eV) and followed physical desorption of the chemisorbed species by a low energy Ar+-ion beam (11.2 eV). By using the optimized chemical adsorption of oxygen-ion on the pristine graphene surface and the optimized physical desorption using Ar+-ion for the graphene ALE process, exactly one monolayer of graphene could be removed per one etch cycle without damaging the graphene surface exposed after the ALE process. Therefore, the graphene layers of the multilayer graphene could be precisely controlled by the number of ALE cycles. One monolayer graphene removal by one graphene ALE cycle could be confirmed not only by the Raman data  but also by measuring optical transmittance of the etched graphene layers . The comparison of AFM images and Raman spectroscopic data of the bilayer graphene before and after the ALE showed uniform thickness control without noticeable damage on the etched graphene surface. We believe that this ALE technique can be very useful not only for next generation graphene-based nanoelectronic devices but also for other nanoelectronic devices based on 2D materials requring the precise layer control.To precisely control the graphene layers without inducing damage, we introduced a graphene ALE process, a cyclic etch process composed of chemical adsorption by a low energy oxygen (OFigure S1, Figure S2, Figure S3, Figure S4


Coupling spins of molecular magnets to two-dimensional (2D) materials provides a framework to manipulate the magneto-conductance of 2D materials. However, with most molecules, the spin coupling is usually weak and devices fabricated from these require operation at low temperatures, which prevents practical applications. Here, we demonstrate field-effect transistors based on the coupling of a magnetic molecule quinoidal dithienyl perylenequinodimethane (QDTP) to 2D materials. Uniquely, QDTP switches from a spin-singlet state at low temperature to a spin-triplet state above 370 K, and the spin transition can be electrically transduced by both graphene and molybdenum disulphide. Graphene-QDTP shows hole-doping and a large positive magnetoresistance ( ~ 50%), while molybdenum disulphide-QDTP demonstrates electron-doping and a switch to large negative magnetoresistance ( ~ 100%) above the magnetic transition. Our work shows the promise of spin detection at high temperature by coupling 2D materials and molecular magnets. et al. show that the spin state of QDTP molecules deposited on graphene and MoS2 couples to their electronic structure, affecting magnetotransport.Engineering a coupling between magnetic molecules and conducting materials at room temperature could help the development of spintronic devices. Loh  However, for an efficient read-out of the spin state of the magnetic molecule, it is important to optimize the magnetoconductance (MC), which is the change in electrical conductance under an external magnetic field, since this parameter characterizes the coupling of the spin state to the local electronic environment3. The electronic coupling between a non-magnetic conductor and the magnetic molecule is used to electrically detect the spin polarization: ideally, this coupling can give rise to a giant magnetoconductance effect that can be electrically transduced. For example, quantum dots directly connected to two metallic or superconducting contacts (source-drain electrode) and capacitively coupled to a third gate electrode, can be used as spin valves; the electrical currents are influenced by the high spin state of the weakly coupled molecular magnet (SMM) crystals physically coupled to the quantum dots5. However, such a device performs only at extremely low temperatures (below 4 K) and hence, these materials have not been applied so far in practical devices8. It is important therefore, to develop magnetic nanomaterials with a room temperature spin state that can be coupled with a suitable electrical conductor via electrostatic, dipolar or flux coupling to detect the spin polarization.Hybrid electronic devices containing magnetic molecules are attractive, as data storage units in quantum information processing, as it enables electric-field controlled spin manipulation9. Recently, Zeng et al. reported a molecule quinoidal dithienyl perylenequinodimethane (QDTP)  Fig. , that, w11. Moreover, 2D atomic layers have also been exploited as non-magnetic spacers in the conventional giant magnetoresistive or tunneling magnetoresistive multilayer devices and have shown greater potential for enhancing spin polarization as compared with oxide films13.Recently, electrical transport experiments conducted at room temperature have identified a number of advantages of using 2D atomic layers, such as long spin diffusion length, large spin signal, and relatively long spin lifetime as compared to metals  or semiconductors 17. In contrast, the effective coupling of room-temperature stable molecular spins and graphene’s magnetoconductance allows one to study spin state-dependent electron transport, which can form the basis of a high-temperature magnetoelectronic device19. On the other hand, MoS2, a semiconducting 2D transition metal dichalcogenide, provides a good platform for studying the role of Coulomb interaction in molecular spin detection. A large electron interaction strength (three orders larger than graphene) due to reduced dielectric screening in semiconducting 2D crystals and the relatively heavy electron band masses of Mo d-orbitals in the presence of high carrier densities indicates that MoS2 should be an ideal system for studying carrier–spin interaction20.Until now, experimental and theoretical investigations of the role of disorder in magnetoconductance have focussed mainly on enhancing the spin-orbit coupling strength of pristine graphene by introducing adatoms, such as heavy metal atoms or atomic hydrogen2. We have chosen graphene as a channel material due to its semimetallic band structure and an intrinsically low-spin-orbit coupling, which allows the study of long spin coherence. The MC at high temperatures (300–390 K) of these two hybrid systems, QDTP-graphene, and QDTP-MoS2, are systematically investigated. Spin transition from the singlet to the triplet state ( ~ 360 K) enhances MC in the hybrid devices. Different charge transfer behavior  is observed for graphene and MoS2 due to the difference in how QDTP molecules bind to these substrates. On graphene, we observe the creation of inhomogeneous charge impurities that lead to an increase in resistance with magnetic field, whereas, the exchange interaction between the triplet spin state of the molecule and MoS2 results in a sharp transition of magnetoconductance ( ~ 100%) at the spin transition temperature.Here we demonstrate a hybrid device comprising of a layer of QDTP molecules, which are charged coupled to 2D materials such as graphene or MoS2 were micromechanically exfoliated from the respective bulk crystals using standard scotch tape method and mounted on degenerately doped silicon substrates with a 300-nm-thick SiO2 overlayer that served as the gate dielectric. The monolayer films were characterized by photoluminescence (PL)  and Hall conductivity (σxy) were measured as a function of temperature (T) and magnetic field (B) using Physical Property Measurement System from Quantum Design (PPMS). Applying a gate voltage Vg, to the Si substrate controlled the charge density in the active channel (graphene or MoS2) device.Monolayers of graphene or MoS2  of pristine graphene field effect transistor, which reveals an ambipolar behavior with Dirac point at Vg = 1.0 V. A positive shift (ΔVg ~ 17 V) of the charge neutrality point (VD) after the deposition of QDTP indicates p-type doping by the molecule  can be fitted well by considering effective medium theory (EMT)14 and using fitting parameters such as carrier mobility μ, and fluctuation of carrier density nr.m.s., which are used to calculate the Boltzmann–RPA conductivity due to screened Coulomb scattering. This Boltzmann–RPA conductivity σB enters into zero magnetic field EMT equation P is a Gaussian distribution centered at an average carrier density n0 with an r.m.s. fluctuation given by nr.m.s. For QDTP-graphene hybrid, we have used an additional parameter, i.e., the short-range conductivity σS for Boltzmann–RPA conductivity, which captures the saturation of conductivity at high carrier density. The carrier mobility 2 V−1 s−1) also shows qualitatively similar behavior (mobility of hybrid device μ ~ 6400 cm2 V−1 s−1) after deposition of QDTP molecules  of pristine MoS2 device at room temperature  rises slowly with increasing back gate voltage, followed by a rapid rise in current as the Fermi level approaches the bottom of the conduction band. For the hybrid device, doping with QDTP leads to an enhancement of σ(Vg) by a factor of three from its pristine value (σDoped ~ 0.10 e2/h vs. σPristine ~ 0.03 e2/h at Vg = 0). Qualitatively similar effect occurs (σDoped ~ 0.06 e2/h vs. σPristine ~ 0.02 e2/h at Vg = 0) for other device which has lower mobility. Inspite of the fact that the conductivity of both pristine MoS2 and MoS2-QDTP hybrid is less than A is the global parameter, β is the critical exponent, and V0 is local parameter. From the difference of n = (267±1)1010 cm−2 ) at different temperatures (T) at a constant perpendicular magnetic field (B = 2 T). Increasing the temperature to 390 K, the Dirac point gradually shifts to 28 V, making graphene more p-doped  as a function of magnetic field (B) for the temperature window 300–390 K is shown in Fig. σxx ~ −0.1 μS) and temperature-independent24  show two distinct features: First, the zero-field conductivity decreases (ΔσxxǀB=0 = −25 μS) when the temperature is increased to 390 K, and second, as a function of the B-field, σxx decreases as temperature increases, as shown in Fig. σxx from its RT value, measured at B = 2 T. In addition, Fig. B-field dependent longidinal conductivity ratio (σxx/σxx(B = 0)) of the hybrid device, averaged over 10 samples, measured at the Dirac point at T = 390 K (σxx/σxx(B = 0) at room temperature in Supplementary Fig. The Hall measurement allows us to study the gate modulation of transverse resistance  at B = 2 T is plotted in Supplementary Fig. xy of pristine MoS2 is temperature independent  of MoS2–QDTP hybrid device as a function of magnetic field (B) for the same temperature window as graphene hybrid is shown in Fig. 2, no change in σxx (B) is observed as the temperature is increased up to 400 K −σxx(0)], is slightly negative (−2 µS) at 330 K, but increases to 7 µS at 400 K. The observed increase in zero-field σxx with temperature is consistent with the Hall conductivity (σxy) measurement. The magneto-conductivity (σxx/σxx (B = 0)), averaged over 10 samples, is plotted as a function of B-field sweep (0 to 200 mT) at different temperatures from 300 to 400 K in Fig. 2 is evident in all the devices, except for the fact that in the case of MoS2 hybrid, we observe positive magneto-conductance.In the case of MoS25, the magnetoresistivity (or magnetoconductivity) of such system has been analyzed previously using a semi-empirical equation25. Herein, the experimental magnetoconductance at Dirac point for each temperature of our graphene-QDTP hybrid is fitted to a modified semi-empirical equation xx,0, low-field conductivity σxx,1, and mobility μ. The extra conductivity term  in the equation accounts for the imbalance in the electron and hole concentrations, unequal electron and hole mobilities, and deviation of sample geometry from the ideal Hall bar26. Above the transition temperature (370 K), σxx,0 (3.36 e2/h) is much larger than σxx, 1 (1.53 e2/h) indicating that magnetoconductivity is probably better described by the unusual [1 + (µB)2]−1/2 dependence at higher temperatures . The temperature dependence of carrier density due to thermal smearing predicted by theory is very small, and should not play an important role in the observed temperature dependence extracted by aforementioned method. This suggests a non-trivial role played by the molecule in carrier activation.QDTP-doped graphene can be considered as an impurity-doped system having an inhomogeneous distribution of electron and holes near the Dirac pointperature 70 K, σx2, the normalized carrier density (nexp/n300 K), averaged over 10 samples, given by two channel model n400K = 1.2 × 1010 cm−2), with a sharp change around 360 K . In slightly electron/hole doped regions, the change in MC is much lower even though a similar trend is observed , the density of Coulomb impurities increases due to higher charge transfer with the triplet state of the molecule27. The MC measured in this hybrid device is relatively large ( ~ 50%) in comparison with other disordered-graphene devices ( ~ 20%)30, and graphene based vertical spin valves ( ~ 15%)32.To investigate the origin of high temperature magnetoconductivity in the case of graphene, the gate–voltage dependence of the magnetoconductance , which should originate from the intrinsic electrical characteristics of the two materials.Our electrical transport measurements reveal a strong change in the MC of the hybrid devices at RT vs. 370 K, which may have its origin in the temperature-dependent charge transfer interactions of the respective 2d materials with QDTP molecules. Interestingly, graphene and MoS2 with QDTP, first-principles calculations were performed to understand the charge transfer behavior in these hybrid systems. QDTP has three conformational isomers or conformers , as shown in Fig. 2 and the adsorbed QDTP molecule. For graphene, the differential charge density analysis shows that the CF3 QDTP accepts a larger number of electrons in the triplet state (0.44e per molecule) than in the singlet state (0.29e per molecule)  in MoS2. It is well known that sulphur vacancies are ubiquitous in MoS2 and can reach concentrations as high as 1010 cm−2 33. These defects have unpaired spins and thus have a stronger interaction with spin molecules. Vs defects also act as charge traps in MoS2, and can be passivated by adsorbates33, one signature of this is the enhanced photoluminescence  adsorption has a much larger charge transfer than that of the singlet-state (0.16e per molecule) adsorption.To understand the origin of the differences in the transport properties of hybrid devices of graphene or MoSle) Fig. . A simille) Fig. –14. The 2 has its origin in the different interactions of these 2D layers with the functional groups in the QDTP. The isosurface plot of the differential charge density reveals that the dicyano groups in QDTP act as electron acceptors with respect to graphene  group located in the in the mid-section of the molecule acts as an electron donor when present on the surface of MoS2 localization2 hybrid, we can consider the magnetic scattering mechanism as reported in diluted magnetic semiconductors (DMS)38, together with spin-imbalance caused by the initialization process of randomly oriented spins40. The exchange coupling between the spin triplet state in QDTP and the 2D channel electrons scales linearly with Wigner Seitz radius rs41. However, for 2D materials, the value of rs depends on both intrinsic factors such as the Fermi velocity, and extrinsic factors like the dielectric screening of the substrate14. For the carrier densities relevant to our experiment, we estimate that for graphene, rs ~ 1, while for MoS2, rs ~ 1000. We attribute the negative magnetoresistance (positive MC) of MoS2 beyond the triplet transition to Ruderman–Kittel–Kasuya–Yosida (RKKY) coupling of the spin triplet state of QDTP to electron spin in MoS2. This is analogous to the coupling between Mn spin states and Ga electrons in the dilute magnetic semiconductor As42. Accordingly, the exchange interaction (J) between the high temperature spin triplet state of the QDTP molecule (Si at site i) and the spin of the carrier in MoS2 (s) is given by the Hamiltonian, 43, which can be reduced by B-field induced alignment of spins, and can be expressed in a simple form as43: A(T) as temperature dependent fitting parameter which has the same qualitative behavior as temperature dependent magnetic signal data (χT) from the SQUID measurement (or square of the magnetic moment) of the QDTP molecule10 . The theoretical values of J based on the current structural model is estimated to be around 131 eV Å−2, which is close to the value (150 eV Å3) reported in As37. We have also estimated J to be 200 eV Å−2 from the experiment following Supplementary Eq. J) and the spin imbalance prevalent in the QDTP molecule, which is enhanced at high temperature due to the spin flip of the molecule 36 or Gd-doped Eu-chalcogenides38, the positive MC effect in QDTP-doped MoS2 at high temperatures is much larger ( ~ 100%).To understand the positive MC in case of the MoSe10 Fig. . The cou2, spin triplets of the adsorbed molecule at high temperature are coupled to charge carriers in MoS2, and an alignment of the non-interacting individual spins with the magnetic field enhances the electron transport. Since the interaction parameter rs in graphene is much weaker than in MoS2, the effect of exchange interaction with the QDTP molecule is much larger in MoS2. The different magnetoconductance behavior of graphene and MoS2 at the spin transition point of the molecule, which can be exploited to detect the spin flip of the magnetic molecules clearly highlights the importance of using the appropriate conduction channel for different targeted application: semimetallic graphene with a higher carrier density is better tailored for impurity-dominated transport, whereas semiconducting 2d materials such as MoS2 are ideal for detecting spin dependent phenomena.Overall, our results show that magnetic molecules on graphene surface can be treated as charge disorder. The transition from singlet to triplet state at high temperature causes a more resistive magneto-transport, which can be enhanced by applying a magnetic field. In the case of MoS2 FET to small magnetic field ( < 20 mT) by two orders of magnitude. This suggests that modifying 2D materials with magnetic molecules can enhance the MC by orders of magnitude. The advantage of QTDP is that the enhancement is operational at elevated temperatures, thus allowing the sensor to be operated at cryogen-free conditions. Finally, our work suggests that biradical molecules with active spin centers may be good candidates for room temperature hybrid spintronic devices with multi-functional properties, enabling the sensing of magnetic, electrical, optical, and chemical stimuli.Our work also highlights the importance of understanding the charge transfer and magnetic phenomena taking place at these molecule-2D hybrid interfaces. Clearly, QDTP molecule enhances the sensitivity of the MoS2 flake is prepared by mechanical peeling and transferred to heavily doped silicon with a layer of 300 nm thick SiO2 (gate capacitance Ci = 11.6 nF/cm2) on top. Optical microscope-based contrast is used to get initial information about the number of layers. To determine the quality and thickness of graphene/MoS2, Raman spectroscopy measurements were carried out. Electrodes were patterned by e-beam lithography using a bilayer resist combination of PMMA and subsequently Ti/Au (3 nm/75 nm) was deposited by an e-beam evaporator. Standard lift-off procedures using warm acetone were followed after the metal deposition. Typical dimension of the channel is as follows: L = 3 µm is the channel length, W = 1.5 µm is the channel width. QDTP molecules dispersed in DCM were drop casted on the devices followed by annealing under vacuum at 400 K to remove the solvent.Graphene or MoS44. Becke88 optimization (optB88) is adopted for the description of the van der Waals interaction between the physisorbed QDTP and graphene/MoS2 by using a kinetic energy cutoff of 400 eV. Here we consider a single QDTP molecule adsorbed in supercells of graphene and MoS2 containing total atoms of 217 and 183, respectively, with a vacuum layer with a thickness of 15 Å. The first Brillouin zone is sampled with a 2 × 3 × 1 Monkhorst–Pack grid. All the structures are fully optimized by minimizing the forces on each atom less than 0.005 eV Å−1. The interfacial charge transfer is obtained via calculating the differential charge density Δρ(r). The amount of transferred electron up to z point in the normal direction is obtained using ρ(z) is the plane-average differential charge density through integrating the Δρ(r) in the x–y plane.First-principles calculations based on spin-polarized density functional theory are performed by using Vienna ab initio simulation packageAll relevant data are available from the corresponding authors.Supplementary InformationPeer Review File


We perform first principle calculations based on density functional theory to investigate the effect of the adsorption of core-excited organic molecules on graphene. We simulate Near Edge X-ray absorption Fine Structure (NEXAFS) and X-ray Photoemission Spectroscopy (XPS) at the N and C edges for two moieties: pyridine and the pyridine radical on graphene, which exemplify two different adsorption characters. The modifications of molecular and graphene energy levels due to their interplay with the core-level excitation are discussed. We find that upon physisorption of pyridine, the binding energies of graphene close to the adsorption site reduce mildly, and the NEXAFS spectra of the molecule and graphene resemble those of gas phase pyridine and pristine graphene, respectively. However, the chemisorption of the pyridine radical is found to significantly alter these core excited spectra. The C 1s binding energy of the C atom of graphene participating in chemisorption increases by ∼1 eV, and the C atoms of graphene alternate to the adsorption site show a reduction in the binding energy. Analogously, these C atoms also show strong modifications in the NEXAFS spectra. The NEXAFS spectrum of the chemisorbed molecule is also modified as a result of hybridization with and screening by graphene. We eventually explore the electronic properties and magnetism of the system as a core-level excitation is adiabatically switched on. K and Advances in 2D physics were stimulated with the discovery of graphene in 2004 . Graphenent past ,4,5,6. Gmi level . This remi level ,7,8, themi level ,10 and hctronics ,12,13. Hctronics ,15. Sevectronics ,17, hetectronics ,19,20,21ctronics ,24,25,26ctronics ,29,30,31Surface science techniques are very apt to the study of these hybrid interfaces, including experimental methods, which involve core excitation of the organic molecule. Among these, X-ray Photoemission Spectroscopy (XPS) and Near Edge X-ray Absorption Fine Structure (NEXAFS) are element sensitive and give information regarding the chemical bonding environment, preferential adsorption orientations of the molecular layer exploiting the dependence of spectra on the direction of the incoming photon field in relation with the transition dipole moments ,33,34,35Density Functional Theory (DFT) ,39,40 haMost of the previous studies relating to organic molecules/graphene interfaces focus on the electronic and magnetic properties of the system in the ground state or with valence excitations ,49. Howez direction orthogonal to the surface plane is 15 Å. The plane wave kinetic energy cutoff is converged to 42 Ry, and those on the energy and force are We perform first principle calculations based on Density Functional Theory (DFT) ,39,40 wif 2.46 Å .For calculating the NEXAFS spectrum, one needs to explore the transitions of the core excited electron to unoccupied bound states. This is simulated by considering a fractional core hole ) pseudopotential, which is better suited to describe the synergy of the core-excited electron with the hole left behind. This model has been introduced in the transition potential approach ,43 and h) method . We cons) method ,37.We start by describing the adsorption configurations of the two selected organic moieties on graphene: Pyridine  of the core electrons, in our case most importantly, that of the C 1s. Values of BE are sensitive to the local bonding environment and therefore rich in chemical information. Theoretically, the BE is defined as the energy difference between the system in the ground state and the one with the core electron removed, i.e., in the presence of a core hole. Absolute BEs are difficult to address numerically, but the most important information is often contained in the relative BEs of atoms of the same species, that is the Core Level Shifts (CLS) with respect to a reference value. Their determination can proceed straightforwardly by electronic structure calculations based on all-electron models, but also on more widespread pseudopotential codes. Indeed, it is sufficient to evaluate the difference in energy of the system, when a modified pseudopotential including a 1s core hole (FCH) replaces the standard one at particular atomic sites . In thisWe can now discuss the effect of core exciting an electron from the C 1s edge of the system and understand the effect of molecular adsorption on the binding energies of the system in terms of the core level shifts. shown in a, which The CLSs for the chemisorbed case of pyridine radical/graphene are shown in dsorbate . Consequtoms see b close tFor the pyridine radical, the CLSs are on average positive, indicating a larger BE than the graphene atoms similar to the case of the physisorbed molecule. However, the difference with the graphene average is now smaller, which implies the core hole at the molecule being more effectively screened by graphene. In this respect, it is helpful to compare the CLSs taken with respect to the molecular average, as we move from the gas phase pyridine molecule to the chemisorbed radical see . In partWe now discuss the Near Edge X-ray Absorption Fine Structure (NEXAFS) spectra calculated for gas phase pyridine and further inspect the NEXAFS spectra for the organic/graphene systems to understand the effect of adsorption on the core excited spectra. The N 1s NEXAFS spectra have been shown in the z axis). Resonances resulting from transitions to states with the x and y photon field directions). These observations are in excellent agreement with previous calculations and experiments [Let us first discuss the C 1s NEXAFS of gas phase pyridine, which is shown in eriments .x and y directions, so that excitation to the molecular LUMO occurs in an experiment with the photon field in the z direction orthogonal to the surface (commonly referred to as p-polarization [We now discuss the effect of physisorption of the pyridine/graphene system on the C 1s NEXAFS spectra shown in rization ).The NEXAFS spectra for the covalently-adsorbed pyridine radical show instead interesting changes with respect to the free molecule, as we report in z (p-polarization). The second one, at about s-polarization) that we compute as the average of the x and y surface azimuths. One can observe by comparing these results to those in We now look at the NEXAFS spectra of graphene and the influence of molecular adsorption on the latter. p-polarized spectrum in p- and s-polarization are very similar, consistent with the sps-polarization are compared. The empty states corresponding to the p-polarization) are perturbed by the orbital missing at the As-polarized spectra is a result of the distortion of the graphene lattice and hybridization of The effect of chemisorption of pyridine radical presents a significant effect on the NEXAFS of graphene. The Abelow EF . The corWe now discuss how the core-level excitation influences the electronic and magnetic properties of the two systems. For the following qualitative analysis, we concentrate on one specific case out of the possible 1s excitation sites; given the large number of inequivalent C atoms that could be considered, we select here the N atom in the molecule. It is instructive to switch on the perturbation adiabatically by performing calculations including a N 1s hole of increasing magnitude ent bond . This unus works ,26,49. TFor a chemisorbed system, in the HCH case, as shown in rity one .Before concluding this section, we remark that the shift of the Dirac cone as induced by a localized perturbation (treated by the repeated supercell approach) is a signature of electrons being attracted towards the perturbation from the surrounding graphene, hence neutralizing the system. Electron transfer from graphene to core-excited molecules has indeed been observed for bipyridine adsorbed on graphene/Ni or Au substrates  where itWe have studied the effect of adsorption of core excited organic molecules on graphene by analyzing the core level shifts and the NEXAFS spectra. For the physisorbed pyridine/graphene case, the relative binding energies of the molecule are comparable with those of the gas phase. One observes that the binding energies of the graphene C atoms close to the adsorption site decreases by ∼0.3 eV when compared to those of farther C atoms. As for the chemisorbed pyridine radical/graphene, the BE of C atom of the molecule participating in the covalent bond increases slightly when compared to the gas phase molecule. The C atom of graphene participating in the chemisorption (AFrom the NEXAFS of adsorbed pyridine, we can conclude that physisorption via weak van der Waals forces minimally alters the NEXAFS spectra of the molecule and is comparable to gas phase pyridine. The same is true for the NEXAFS spectra of graphene, which resembles the one of pristine graphene. As for the chemisorbed pyridine radical/graphene case, we find a significant distortion in the NEXAFS spectra of the molecule and especially graphene atoms close to the adsorption site. Following the increase in binding energy of the C atom of the molecule participating in the covalent bond, its contributions to the NEXAFS spectra also shift to higher energies. The loss of the Upon creation of the core hole, the system gradually reaches a solution, with the LUMO shifting towards the Fermi level. For physisorbed pyridine/graphene, a magnetic moment mostly localized on the molecule is computed. Upon core excitation of the chemisorbed pyridine radical/graphene, the LUMO and the mid-gap state hybridize in the spin-up channel, whereas the spin-down LUMO remains unoccupied and decoupled from graphene mid-gap states. This induces a magnetic moment on the system, which is shared between the molecule and graphene.


To better understand the efficacy and safety of anti-PD-1/PD-L1 therapy  in patients with previously treated advanced non-small-cell lung cancer (NSCLC).The Cochrane Controlled Trial Register, Embase, Medline, and the Science Citation Index were searched for prospective published reports of atezolizumab, pembrolizumab, nivolumab in previously treated patients with advanced NSCLC.Finally, we identified 14 prospective published reports including four trials of atezolizumab covering 542 subjects, three trials of pembrolizumab covering 1566 subjects, seven trials of nivolumab covering 1678 subjects. When compared to docetaxel, anti-PD-1/PD-L1 therapy could significantly improve overall survival  and progression-free survival  for previously treated patients with advanced NSCLC. Anti-PD-1/PD-L1 therapy produced an overall response rate of 19% in the 2374 evaluable patients. When using docetaxel as the common comparator, indirect comparison of anti-PD-1/PD-L1 therapy versus EGFR-TKIs showed progression-free survival benefit  and overall survival benefit  for those patients with EGFR wild-type. Meanwhile, for those EGFR mutant patients, indirect comparison indicated that anti-PD-1/PD-L1 therapy was inferior to EGFR-TKIs therapy in terms of progression-free survival , but no survival difference .Anti-PD-1/PD-L1 therapy could produce progression-free survival and overall survival improvement over docetaxel for patients with previously treated NSCLC. For EGFR wild-type patients, anti-PD-1/PD-L1 therapy seemed to prolong progression-free survival and overall survival when compared to EGFR-TKIs. Meanwhile, for these EGFR mutant patients, anti-PD-1/PD-L1 therapy was inferior to EGFR-TKIs therapy in terms of progression-free survival. The interaction of programmed death 1 (PD-1) with the non-small-cell lung cancer (NSCLC) expressed ligands programmed death-ligand 1 (PD-L1) and PD-L2 could downregulate T cell activity and promote tumor immune escape . RecentlEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and erlotinib have been used as suggested for heavily pretreated molecularly selected patients with NSCLC –22. BothFinally, we identified 14 prospective published reports including four trials of atezolizumab covering 542 subjects, three trials of pembrolizumab covering 1566 subjects, seven trials of nivolumab covering 1678 subjects. And, four trials of EGFR-TKIs covering 2475 subjects were also included for indirect comparison . All RCTI2=0%, P=1.00 for OS analysis; I2=44%, P=0.13 for PFS analysis. The HRs in this analysis of OS favored anti-PD-1/PD-L1 therapy across most prespecified subpopulation; the exceptions were the subpopulation who lived in the rest-of-the-world geographic region, those with age more than 75 years, those with central nervous system metastases, those who had never smoked, and those with EGFR mutation, and prolonged the PFS by 17% Figure . The valSubgroup analyses by the tumor PD-L1 expression level indicated that anti-PD-1/PD-L1 therapy could significantly prolong both PFS and OS in patients of high PD-L1 expressions, but not in those with low expressions, regardless of PD-L1 expression level of 1%, 5%, and 10% Figure . FurtherGenerally, the rates of adverse events (AEs) of anti-PD-1/PD-L1 therapy seemed to be lower than that of docetaxel Figure .When using docetaxel as common comparator, our indirect comparison indicated that anti-PD-1/PD-L1 could reduce the progression of risk by 38% , and prolonged OS by 40%  for those EGFR wild-type patients. These trials were enrolling patients using different biomarker cutpoints, and which cutpoint could best predict the activity of anti-PD-1/PD-L1 therapy still remains undefined. Furthermore, this analysis indicated statistically significant difference between PD-L1 expression level of≥5% and <5% (P=0.0008), between PD-L1 expression level of≥10% and <10% (P=0.005) in OS analysis. And, a marginal difference between PD-L1 expression level of≥5% and <5% (P=0.05) was shown in PFS analysis. Analysis by smoking history indicated favorable PFS and OS outcomes in former and current smokers, which could be explained by the expected higher mutational load in smoking-associated lung cancer. And there were separate anti-PD-1/PD-L1 therapy studies for squamous and non-squamous, this analysis indicated that anti-PD-1/PD-L1 therapy could provide similar benefit for squamous and non-squamous NSCLC.In this pooled analysis, an impressive ORR of 19% derived from anti-PD-1/PD-L1 therapy compared favorably to the ORRs of 7% to 9% from current second-line therapies for advanced NSCLC –26. FurtClinicalTrials.gov number NCT02220894). This study is enrolling patients using distinct biomarker cutpoints, and the final analyses could provide help to determine which cutpoint best predicts activity of anti-PD-1/PD-L1 therapy monotherapy in these earlier lines of therapy. For those patients for whom anti-PD-1/PD-L1 therapy monotherapy is not as effective as cytotoxic chemotherapy, in combination with chemotherapy [Whether the benefit of anti-PD-1/PD-L1 therapy extends to patients with a tumour proportion score of less than 1% needs to be defined in future trials. KEYNOTE-024 trial  had indiotherapy  or otherotherapy  might beResponses with anti-PD-1/PD-L1 therapy were seen in patients with EGFR- and KRAS-wildtype and EGFR- and KRAS-mutant NSCLC; however, low numbers of enrolled patients in these trials precluded relationship of mutation status with clinical outcomes after anti-PD-1/PD-L1 therapy. Futhermore, the introduction of anti-PD-1/PD-L1 therapy and EGFR-TKIs for heavily pretreated patients with NSCLC had also created a dilemma regarding whether anti-PD-1/PD-L1 agent was better than EGFR-TKIs, or vice versa. Using the common comparator of docetaxel, our indirect comparison indicated that PFS and OS with anti-PD-1/PD-L1 were superior to that with EGFR-TKIs. Meanwhile, for those EGFR mutant patients, anti-PD-1/PD-L1 therapy was inferior to EGFR-TKIs therapy in terms of PFS.I2=44%) and OS  analysis, high and significant for ORR  analysis  They dealt only with previously treated advanced NSCLC patients. (2) They enrolled patients treated with anti-PD-1/PD-L1 therapy or EGFR-TKIs.(3) Acceptable comparator was docetaxel. (4) They could provide data about adverse events rate, response rate, overall survival (OS) and (or) progression free survival (PFS). (5) These studies are prospective. Multiple reports about a single trial were considered as one. All potential trials were reviewed by two investigators separately (Y.X.Z and Z.X.S.).Two reviewers (Y.X.Z and Z.X.S.) independently assessed the quality of selected RCTs using the following criteria: (1) generation of allocation sequence, (2) description of dropouts, (3) masking of randomization, intervention, outcome assessment, (4) intention-to-treat analyses. Each criterion was rated as yes, no or unclear.The primary objective was to define the Hazard Ratios (HRs) of PFS and OS for anti-PD-1/PD-L1 therapy versus EGFR-TKIs, and calculate ORR from anti-PD-1/PD-L1 therapy. The secondary objectives were to evaluate the Risk Ratio (RRs) of adverse events for anti-PD-1/PD-L1 therapy versus docetaxel.MA is the estimate of direct comparison between intervention anti-PD-1/PD-L1 therapy vs. docetacel and EGFR-TKIsMA is the direct comparison of intervention EGFR-TKIs vs. docetacel, then the estimate of the adjusted indirect comparison of intervention EGFR-TKIs vs. docetacel (such as log HR) is estimated by anti-PD-1/PD-L1 therapyMA-EGFR-TKIsMA [MA) + Var (log (EGFR-TKIsMA) [The indirect meta-analysis preserves the randomization within a RCT meanwhile, combines all available comparisons between treatments , 24. TheR-TKIsMA , 24. Bec-TKIsMA) , 24.2 test and quantified with the I2 statistic. Statistical heterogeneity was considered significant when the test produced a P-value <0.1. The I2 statistic was calculated as a measure of the degree of heterogeneity among selected studies, where I2 values of 25%, 50% and 75% were considered low, moderate and high degrees of heterogeneity respectively. We also undertook subgroup analyses to sought the source of heterogeneity. Publication bias was evaluated with Egger's test. All meta-analyses were undertaken with Review Manager  and Stata ver. 12.0 software . Statistical significance was defined as a P value of less than 0.05 except for heterogeneity test.All these analyses were undertaken using a random-effects model which could provided a more conservative result. The heterogeneity among these trials was evaluated using Cochrane χ


Gliomas are the most common type of primary brain tumor in adults. High-grade neoplasms are associated with poor prognoses, whereas low-grade neoplasms are associated with 5-year overall survival rates of approximately 85%. Despite considerable progress in treatment modalities, the outcomes remain dismal. As is the case with many other tumors, gliomas express or secrete several immunosuppressive molecules that regulate immune cell function. Programmed death-ligand 1 (PD-L1) is a coinhibitory ligand that is predominantly expressed by tumor cells. The binding of PD-L1 to its receptor PD-1 has been demonstrated to induce an immune escape mechanism and to play a critical role in tumor initiation and development. Encouraging results following the blockade of the PD-1/PD-L1 pathway have validated PD-L1 or PD-1 as a target for cancer immunotherapy. Studies have reported that the PD-1/PD-L1 pathway plays a key role in glioma progression and in the efficacy of immunotherapies. Thus, progress in research into PD-L1 will enable us to develop a more effective and individualized immunotherapeutic strategy for gliomas. In this paper, we review PD-L1 expression, PD-L1-mediated immunosuppressive mechanisms, and the clinical applications of PD-1/PD-L1 inhibitors in gliomas. Potential treatment strategies and the challenges that may occur during the clinical development of these agents for gliomas are also reviewed. Gliomas account for 51.4% of all primary brain tumors and are thus the most common primary brain tumor in adults . The abiThe success of immunotherapy in other cancers and the current understanding of the interaction between tumors and the immune system have generated increasing interest in the use of PD-1/PD-L1 inhibitors in the treatment of gliomas, particularly glioblastomas (GBMs). The first large phase III trial of NIVO in patients with GBM  was initiated in January 2014 and is ongoing. Given that the PD-1/PD-L1 pathway is critical for downregulating the immune responses of gliomas, we reviewed its expression, the mediated immunosuppressive mechanisms, and the clinical applications of PD-1/PD-L1 inhibitors. We also considered potential treatment strategies and the challenges that may occur during the clinical development of these agents in gliomas.The expression of PD-L1 has been detected in glioma cell lines and tumor tissue. The expression of PD-L1 was detected in glioma cell lines as early as 2003 by Wintterle et al. . They foP = 0.001), and this finding may be related to the invasion of gliomas. Upregulation of PD-L1 at the edge of the tumor forms a barrier between the tumor cells and cytolytic T cells; this phenomenon has been termed a “molecular shield” and contributes to the high rate of malignant infiltration and the escape from immune surveillance during invasion into the adjacent brain tissue.Recent studies have investigated the distribution of PD-L1 expression in glioma tissues. The patterns of PD-L1 expression were described as two main staining patterns: diffuse/fibrillary patterns and membranous patterns. Further analyses revealed no significant difference in the extent of diffuse/fibrillary or membranous PD-L1 expression between newly diagnosed and matched recurrent glioblastoma specimens . Yao et P < 0.001, χ2 test). In another study, in which 48 patients with gliomas were enrolled, western blot analyses revealed a significantly higher level of PD-L1 expression in HGGs (n = 24) than in LGGs  [To date, all of the relevant studies have demonstrated that the expression of PD-L1 in tumor tissues is correlated with glioma grade, which demonstrates that PD-L1 may be a candidate tissue biomarker for gliomas. Wilmotte et al.  found th< 0.001) . Baral e< 0.001)  reportedP = 0.023). The patients with high PD-L1 expression levels (dichotomized into low and high at the cut-off point 0.37) exhibited a significantly increased risk of death compared with patients with low expression levels (P = 0.0231), and PD-L1 was an independent factor that was negatively associated with survival (P = 0.0343). The prognostic influence of PD-L1 expression at the protein level was evaluated in a survival analysis of 94 GBM samples. Using the median as the cut-off point, the patients with >2.77% PD-L1-positive cells exhibited a trend toward worse OS (P = 0.066). However, when a cut-off of 5% positive cells was used, as has been used in numerous other studies of solid malignancies, high expression was associated with a significantly shorter survival (P = 0.0086), which confirmed the results obtained with the mRNA data. The different PD-L1 expression threshold levels and assay technologies  likely contributed to these inconsistent conclusions [Major research efforts have been made to evaluate the prognostic value of PD-L1 in gliomas. In a study of 229 glioma (grades I–IV) patients, Zeng et al.  found noclusions . TherefoGliomas have long been recognized as immunosuppressive neoplasms that are characterized by the activation of various immune escape mechanisms. The coinhibitory characteristics of the PD-L1 molecule are attributed to the binding of this molecule to its receptor, PD-1, on tumor-specific T cells. This binding leads to apoptosis of the tumor-specific T cells and subsequently provides an immune escape for glioma cells that is similar to that of several types of extracranial tumors, including melanoma, lung cancer, gastric cancer, Ewing sarcoma, and head and neck cancer , 22. TheThe upregulation of PD-L1 on tumor cells plays a significant role in the immune escape mediated by gliomas. The mechanisms of the upregulation of PD-L1 in gliomas are illustrated in Fig. Constitutive oncogenic signals are shown to mediate intrinsic induction of PD-L1 as an “innate resistance” mechanism of immune evasion. This is evidenced by the small fraction of human cancers that lack TILs in the tumor microenvironment but still express high levels of PD-L1 , 31. ParRecent findings have supported the notion that PD-L1 upregulation in tumor cells is related to both innate and adaptive resistance mechanisms. Han et al.  found thThe blockade of PD-1/PD-L1 can elicit effective anti-tumor T cell responses. In the past 5 years, the targeting of the PD-1/PD-L1 axis has been at the forefront of immunotherapy due to its remarkable clinical efficacy in melanoma and non-small cell lung cancer clinical trials , 45. TheMost of the preclinical research into gliomas involved targeting the PD-1/PD-L1 axis in addition to other immunosuppressive inhibitors. Huang et al.  reportedP < 0.001) compared with 27 days in the RT-alone group and 30 days in the anti-PD-1 monotherapy group, and 15 to 40% of the mice became long-term survivors (>90 days after implantation). On day 21 after implantation, infiltration by cytotoxic T cells had increased and regulatory T cell levels had decreased in the combined treatment group compared with the single-modality arms. T cell immunoglobulin mucin-3 (TIM-3) is another immune checkpoint molecule and acts as a negative regulator of the immune system [P < 0.05) compared with the other treatment arms. This triple-modality treatment increased the infiltrations of IFN-γ+ and tumor necrosis factor-α+ CD4 T cells as well as IFN-γ+ CD8 lymphocytes into the tumor. These findings indicate that anti-PD-1/PD-L1 pathway treatment combined with SRS may be a feasible treatment strategy in gliomas.Combination treatment with a PD-1/PD-L1 inhibitor and radiotherapy (RT) is an attractive option given the potential for increased tumor antigen release and presentation. RT counteracts the immunosuppressive tumor microenvironment by enhancing the presentation of normally suppressed tumor-associated antigens, increasing the expression of major histocompatibility complex class I and proinflammatory cytokines, promoting dendritic cell maturation and downregulating Fas ligand expression. RT can foster dendritic cell maturation and promote CD8+ T cell recruitment into tumors . In prece system . Kim et e system  evaluateBevacizumab is a humanized mAb that targets vascular endothelial growth factor and became the third drug approved by the FDA for use in recurrent GBM in 2009 . HoweverClinical trials have been initiated to determine the potential of PD-1/PD-L1 checkpoint inhibitors as monotherapies and combination therapies for glioblastomas Table .Table 2SNIVO is a fully human IgG4 subtype antibody that targets human PD-1 with a stabilizing hinge region mutation that is resistant to the exchange of IgG4 molecules. The FDA has approved NIVO for unresectable or metastatic melanomas and NSCLCs. NIVO is currently being investigated as a monotherapy for GBM in a phase II trial (NCT02550249). Ongoing trials of NIVO in combination with galunisertib (a kinase inhibitor of TGFβRI) are being conducted in patients with GBM (NCT02423343). Ipilimumab (IPI) is a fully humanized mAb against CTLA-4 and was approved by the FDA in 2011 for advanced melanoma –60. BasePembrolizumab is a humanized monoclonal IgG4 anti-PD-1 antibody consisting of a high-affinity mouse anti-PD-1-derived variable region grafted onto a human IgG4 immunoglobulin molecule with an engineered Fc region for stabilization. Pembrolizumab was approved in 2014 by the FDA for the treatment of patients with IPI-treated advanced melanoma . In a reIn addition to the PD-1 inhibitors discussed above, there are two anti-PD-L1 agents that are currently being evaluated in clinical trials for gliomas. The human anti-PD-L1 antibody MEDI4736 has demonstrated a durable response in patients with melanoma and NSCLC , and MEDn = 3) and nausea (n = 3). For the N1 + I3 group, the adverse events included fatigue (n = 8), diarrhea (n = 7), increases in glutamic oxalacetic aminopherase and lipase (n = 5 each), increases in vomiting and alanine aminotransferase (n = 4 each), and amylase increase, headache, hyperthyroidism, nausea, and maculo-papular rash (n = 3 each). Among these N1 + I3 patients, 8/10 developed grade 3–4 adverse events. Treatment discontinuation due to drug-related adverse events, which included colitis, cholecystitis, diabetic ketoacidosis, confusion, and increased lipase, occurred in only 5 patients with N1 + I3. Updated results regarding adverse events were presented at the 2016 ASCO Annual Meeting [n = 8), anorexia (n = 1), dermatological issues (n = 6), arthralgias (n = 2), and endocrine problems (n = 1). In the NSCLC cohort, the serious adverse events included (one patient each) acute kidney injury (grade 2), pneumonitis (grade 3), colitis (grade 3), hyperkalemia (grade 4), and fatigue (grade 3). The grade 1–2 adverse events included colitis or diarrhea (n = 3), acute kidney injury (n = 1), fatigue (n = 5), anorexia (n = 3), dermatological problems (n = 4), arthralgias (n = 1), endocrine problems (n = 5), and hematologic effects (n = 2). Neurological adverse events were also reported in the clinical trial, and to the best of our knowledge, data regarding PD-1 and PD-L1 inhibitors remain scarce [n = 1), grade 1–2 seizures (n = 3), headache (n = 3), and dizziness (n = 1); in addition, 2 of these patients developed neurologic symptoms due to peri-lesional edema . The neurological adverse events in the NSCLC cohort were grades 1–2 and included cognitive dysfunction (n = 1), headache (n = 4), dizziness (n = 2), and stroke (n = 1). There were no treatment-related deaths or autoimmune events, which indicated the safety of the blocking PD-1 or PD-L1 antibodies in patients with CNS tumors.The aim of antibodies that target either PD-1 or PD-L1 is to block the PD-1/PD-L1 pathway with the goal of adjusting and normalizing immunity to a desirable level while not enhancing immunity in general . The ach Meeting . In the  Meeting . These rn scarce . The neuThere are some challenges to the clinical application of targeting the PD-1/PD-L1 axis as a therapeutic modality in patients with glioma. First and the most important, biomarkers that identify patients who are likely to respond to PD-1 or PD-L1 inhibition have not been defined. Although PD-L1 immunohistochemistry has been approved by the FDA as the only predictive companion diagnostic test for the use of pembrolizumab in NSCLC patients, improved survival outcomes have been observed in many PD-L1-negative patients . AdditioPD-L1 is expressed in glioma cells, correlates with tumor grade, and contributes to immunoresistance. The PD-1/PD-L1 pathway plays an important role in glioma biology. The use of anti-PD-1/PD-L1 antibodies in immunotherapeutic strategies for gliomas is attracting increasing attention. With the goal of improving the efficacy of anti-PD-1/PD-L1 immunotherapy, many studies have focused on combination treatment, including the targeting of multiple immune inhibitors, RT, ablation, chemotherapy, and other molecular targeting therapies. Because there is a need for better and safer treatment strategies for gliomas, the initiation of a greater number of successive clinical trials associated with immune checkpoint blockade along with further exploration into the mechanism of tumor immunity would be warranted.