HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and  •We report the crystal structure of human PD-1 and the mapping of the PD-1 ligand binding site.•A high-affinity variant was identified and used to engineer a soluble PD-1 Ig fusion protein (HA PD-1 Ig) for immunotherapy.•HA PD-1 Ig enhanced T cell activation and strongly synergized with RT to control tumor growth in a lung carcinoma model.PD-1 is an inhibitory immune receptor that dampens T cell responses. PD-1 blockade is a successful strategy for cancer immunotherapy.We report the crystal structure of human PD-1 and mapping of its ligand binding site. A high-affinity mutant (HA PD-1) was identified and used to engineer a blocking Ig fusion protein, expected to disrupt the endogenous PD-L/PD-1 pathway.HA PD-1 Ig increased lymphocyte proliferation and cytokine production. Moreover, in combination with radiation therapy it enhanced control of tumor growth in a Lewis lung carcinoma model. This study offers an alternative strategy to manipulate the PD-1 pathway for tumor immunotherapy. PD-1 was discovered in lymphoid murine cell lines induced to undergo apoptosis by PMA stimulation . Subsequ+ and CD8+ T cells, as well as on B cells, myeloid cells (monocytes and some dendritic cells) and NK T cells, suggesting multiple roles in immune regulation , regulating the development, maintenance and functional response of induced Treg cells, and facilitating de novo conversion of naïve CD4+ T cells to Foxp3+ Tregs  and PD-L2 , which belong to the B7 family and share 34% identity. PD-L1 mRNA is ubiquitously expressed by immune cells, as well as non-hematopoietic cells, and cell surface PD-L1 is upregulated upon activation. Cytokines such as IFN-γ and TNF-α induce PD-L1 expression on T and B cells, endothelial and epithelial cells . PD-L1 oPD-1 and its ligands are single-pass type I transmembrane proteins, similar to other members of the CD28/B7 family . PD-1 co+ T cells in chronic viral infections, such as LCMV , as well as with murine PD-L2  have been determined, and most recently the crystal structure of human PD-1 in complex with human PD-L1 was reported . Notably, in an in vivo model of murine Lewis lung carcinoma, HA PD-1 Ig treatment in combination with radiation therapy (RT), synergistically decreased local and metastatic tumor burden, increased survival and induced immunological memory responses towards tumor re-challenge. These studies demonstrate that our structure-guided HA PD-1 Ig variant, in combination with stereotactic radiotherapy, represents a promising strategy for tumor immunotherapy.Here we report the identification and biochemical, structural and functional characterization of a high-affinity mutant of human PD-1 variant (A132L) exhibiting approximately 45- and 30-fold higher affinities to PD-L1 and PD-L2, respectively. A soluble chimeric Ig fusion construct of this high-affinity PD-1 variant (HA PD-1 Ig) significantly increased proliferation and cytokine production of human T cells in an 22.1E. coli Rosetta 2 (DE3) pLysS strain (Novagen). Wild type and mutant human PD-1 inclusion bodies were purified and refolded as described for mouse PD-1  were cloned into the pET3a vector between the NdeI and BamH1 restriction sites. Expression plasmids were transformed into use PD-1 . The ref2.2P 6522 . Data were integrated and scaled with HKL2000  as the precipitant. Data were collected at the National Synchrotron Light Source (NSLS) beamline X-29A, and were consistent with the hexagonal space group  HKL2000 . The hum HKL2000  and crys HKL2000 . The finPROCHECK . Superpo2.3Mutants of full-length human PD-1 for mammalian expression were generated by PCR-based mutagenesis, using the Gateway pEF5/FRT/V5-DEST destination vector (Life Technologies). PD-1 constructs were transiently transfected into HEK293 cells using either Lipofectamine2000 (Life Technologies), or calcium phosphate transfection. Residues were selected for mutagenesis based on similarity with mouse PD-1, and analysis of the mouse PD-1/PD-L2 interface reported earlier . Full-le2.4Human and mouse PD-L1 and PD-L2 Ig fusion proteins were purchased from R&D Systems, and characterized by SEC prior to use. Functional grade PD-L1 blocking antibodies was purchased from Affymetrix eBioscience (clone MIH1) and from BioXCell (clone 10F.9G2). Human CD11c-FITC and MHCII-APC antibodies were purchased from Life Technologies; CD14-FITC, PD-1-FITC (MIH4 and J116), PD-L1-PE-Cy7, PD-L2-APC, CD83-APC and B7-1-FITC antibodies from Affymetrix eBioscience.2.5d), a range of concentrations (0.01–100 μM) of PD-1 or its mutants were injected sequentially over the flow cell at a rate of 20 μl/min. Kds were obtained by fitting the data to the non-linear 1:1 Langmuir binding model, using the relationship: R = RmaxC / (C + Kd), where R is the bound analyte in response units (RU), Rmax is the maximum response level, C is the concentration of the free analyte in μM and Kd is in μM.Surface Plasmon Resonance (SPR) binding experiments were performed with a BIAcore 3000 optical biosensor at 25 °C. Murine or human PD-L1-Ig or PD-L2-Ig fusion proteins were immobilized on the surface of a CM5 sensor chip by free amine coupling, followed by deactivation with ethanolamine (following the manufacturer's protocol). For determination of equilibrium dissociation constants (K2.62 +-NTA Agarose (Qiagen). The proteins were eluted with 250 mM imidazole (pH = 8) and exchanged into 1xPBS. The purity and quality of the eluted protein was assessed by SDS-PAGE and SEC.The ectodomain of human PD-1 was fused to the Fc domain of human IgG1 followed by C-terminal 6xHis tag, and the endogenous PD-1 signal peptide was replaced by β2-microglobulin signal peptide. Mutant PD-1 Ig fusion proteins  were generated by PCR-based mutagenesis. The fusion constructs were cloned into the pIRES2-EGFP expression vector (Clontech) and transfected into HEK293 cells using calcium phosphate. Stable cell lines were generated by antibiotic selection  and FACS sorting. Ig fusion proteins were purified from cell culture supernatants by affinity chromatography using Ni2.72, Fcγ fragment specific (Jackson Immunoresearch). After washing, the cells were fixed in 2% paraformaldehyde. Samples were processed using LSRII or FACS Canto II flow cytometers (BD Biosciences), and analyzed using Flow Jo software . Mean Fluorescence Intensities were normalized to wild type PD-1 binding. Cell surface expression of PD-1 constructs was quantified by monoclonal antibody staining.HEK293 cells transiently transfected with wild type or mutant PD-1 constructs were incubated with PD-L1 or PD-L2 Ig fusion proteins (0.01–50 μg/ml) for one hour on ice. After washing, the cells were further incubated for 30 min in the presence of APC-conjugated goat anti-human IgG F(ab′)2 (Jackson Immunoresearch). The expression of the PD-L1 and PD-L2 ligands on the cell surface was verified by monoclonal antibody staining.For binding experiments using human PD-1 Ig proteins, differentiated human dendritic cells, or PD-L1 or PD-L2 transfected HEK293 cells were incubated with the Ig fusion proteins, followed by incubation with Cy5-conjugated goat anti-human IgG F(ab′)2.8+ T cells from PBMCs of healthy donors were purified by negative selection, using EasySep kit (Stemcell Technologies).PBMCs from the peripheral blood of healthy donors were isolated by Ficoll gradient centrifugation. Monocytes were enriched by plastic adherence for 2 h. After removing non-adherent cells, adherent cells were cultured for 5 days in the presence of GM-CSF 100 ng/ml and IL-4 50 ng/ml (R&D Systems) to induce formation of immature dendritic cells. Fresh cytokines were added every 3 days. On day 6 of culture, 100 ng/ml TNF-α was also added to promote the formation of “mature” dendritic cells. These cells were used for subsequent binding and functional assays. Human CD45/well) were co-cultured with the differentiated dendritic cells derived from an allogeneic donor, using 16:1 T cell: DC ratios. Prior to adding to the cultures, DCs were irradiated using 2000 rad, and incubated with various concentrations of wild type and mutant human PD-1 Ig fusion proteins for 30–60 min. After addition of T cells, allogeneic stimulation proceeded for 5 days before addition of 1 mCi/well of 3H-thymidine (Perkin Elmers) for the final 12 h of incubation. Culture supernatants collected at day 4 were assayed for multiple cytokines using FlowCytomix Multiplex kits Th1/Th2 11-plex, or Th1/Th2/Th17 13-plex (Affymetrix eBioscience).For allogeneic stimulation, freshly purified human T cells  cell line was purchased from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco) supplemented with 10% FBS and 250 IU of penicillin/streptomycin.2.105 3LL cells (in 30 μl) subcutaneously into the dorsum of the right hind limb . Primary dorsal hind limb tumors exhibit Gompertzian growth, with a phase I volume of 40–80 mm3, phase II volume of 150–300 mm3, and phase III volume of > 300–500 mm3. Therefore, treatment efficacy was evaluated by determining the tumor growth delay (TGD) to quadruple the initial volume (Ve) according to: te = t1 + (t2 − t1)log(Ve/V1)/log(V2/V1), where V1 and V2 are measured volumes that flank Ve at times t1 and t2, respectively. Tumors ≥ 300–500 mm3 have reached phase III growth due to anatomical and vascular limitations, and below-the-knee amputations were performed on mice for the metastases studies, in accordance with our IACUC approved protocol.Primary tumors were established by subcutaneous injection of 10ind limb A. Once p2.11en bloc and washed with PBS. Lungs were submerged and fixed in Fekete's solution for 24 h, followed by transfer to 4% paraformaldehyde prior to enumeration of pulmonary metastases. The left lung and the 4 lobes of the right lung were isolated and nodules counted under a dissecting microscope.Isolated lungs from mice receiving below-the-knee amputation (as previously described) were injected intratracheally using 1 mL of Fekete's solution  to insufflate the lobes. The trachea was clamped, and the entire lung and heart removed 2.125 3LL tumor). Naïve mice were also injected as controls. Mice were monitored until day 66 and then sacrificed to evaluate immune memory T cell populations  in spleen and draining lymph nodes. The following antibodies were used: CD62L-PE clone MEL-14, CD44-FITC clone IM7 from eBioscience; CD45-Alexa 700 clone 30-F11, CD3-Pe-Cy5 clone 145-2C11, PD-L1-BV421 clone MIH-5 from BD Bioscience; CD4-APC-Cy7 clone RM4-5, CD8-PerCP clone 53–67 from Biolegend. Cells were acquired on a BD LSR-II Flow Cytometer and analyzed using the FlowJo software .Fifty-seven days after tumor treatment, cured mice were reinjected on the opposite foot with the same number of cells used for the first tumor inoculation (1 × 102.13http://www.slicer.org). For radiation delivery, a 1 mm copper filter was fitted with a 10 × 10 mm collimator at a source-skin distance (SSD) of 35 cm, energized at 220 kV and 13.0 mA. Mice were anesthetized using a continuous flow of 1.5% isoflurane in pure oxygen at a rate of 1.5 L/min. Mice were placed in a left lateral decubitus position on the stage, attached to a motorized platform, and the tumor-bearing right hindlimb was extended, elevated, and secured to a 1.5 cm adhesive platform to minimize extraneous tissue exposure. 20 Gy was delivered over each of three successive days for a total hypofractionated dose of 60 Gy.All radiation was delivered using Xstrahl's Limited Small Animal Radiation Research Platform (SARRP). Briefly, a motorized stage and a constant voltage 225 kV X-ray tube, mounted on a motorized gantry, were controlled via a SARRP desktop application. Treatment plans were designed with 5 degrees of motion: translation  and rotation of animal stage, and by independent gantry rotation. Image-guided radiation therapy (IGRT) was performed using the SARRP's on-board cone beam CT (CBCT). CBCT imaging is achieved using a 1 mm aluminum filter with 50 kV, 0.7 mA, and a 20 × 20 cm flat panel detector for acquisition. Following CBCT acquisition, the treatment plan was constructed using 3D Slicer, an open-source software package (Mice were randomly distributed into the following treatment groups: 1) Control group receiving i.p. injection of 0.2 mg human IgG isotype control every 3 days for a total of 5 doses; 2) HA PD-1 Ig group, 3) mPD-L1 Ab group, injected i.p. with 0.2 mg HA PD-1 Ig, or mPD-L1 Ab, respectively, every 3 days for a total of 5 doses; 4) RT group treated with 3 days of successive 20 Gy fractions followed by 0.2 mg human IgG isotype i.p. 3 days after the final 20 Gy fraction, for a total of 5 doses; 5) Combination RT + HA PD-1 Ig, or 6) RT + mPD-L1 Ab groups treated with 3 days of successive 20 Gy fractions followed by i.p. injection with 0.2 mg HA PD-1 Ig, or mPD-L1 Ab, respectively, 3 days after the final 20 Gy fraction for a total of 5 doses.2.14t-test. Data were tested for normality and variance. A p value of < 0.05 was considered significant. All experiments were performed at least twice for reproducibility.Data were analyzed using Prism 5 software (GraphPad Software Inc.). For the analysis of three or more groups, the non-parametric ANOVA, followed by post-test comparisons , was used. Analysis of differences between two normally distributed groups was performed using the Student's 2.15All animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Albert Einstein College of Medicine.33.1Based on our previously reported crystal structure of the mouse PD-1/PD-L2 complex , and seqConsistent with earlier studies on mouse PD-1 , mutatioThe general similarity of the human PD-1 mutants presented here and the murine PD-1 mutants reported earlier is not surprising, as 14 of the 17 residues that contribute to the binding interfaces are conserved between mouse and human PD-1, with 9 residues being conserved in all species  exhibits typical IgV domain topology, with the front β-sheet (strands A'GFCC'C″) and back β-sheet (strands ABED) forming a two layered β-sandwich stabilized by the canonical disulfide bond between the B (C54) and F (C123) β-strands (α atoms), however, the presence of the C″ strand could not be modeled in the human PD-1 structure due to lack of electron density for that region. Subsequent to deposition of our structure, the solution NMR structure of wild type human PD-1 was reported, which exhibits a high degree of similarity, with an RMSD of 1.4 Å over 101 Cα atoms  complex  shows liSuperposition of the human PD-1 structure with the mouse PD-1/mouse PD-L2 complex  predicts that Y68 in human PD-1 contributes to a hydrophobic cluster including W110, Y112, and Y114 on the G strand of PD-L2 E and G. 3SBW) and mouse PD-L2 (pdb ID 3RNK and 3BP6). Although there were no major differences in stoichiometry, or overall conformation between wild type and A132L PD-1 complexes, as predicted, the Leu residue present in the mutant made a greater number of interactions than the Ala residue in wild type PD-1 with PD-L1 and PD-L2, consistent with its higher affinity (data not shown).The A132L mutation in human PD-1 is predicted to afford additional van der Waals interactions with Y56, I54 (C strand) and M115 (F strand) in human PD-L1 F. Simila3.3on and koff rates, similar to those observed earlier for the PD-1/PD-L interactions for mouse PD-1  . PD-L1 ouse PD-1 . Fittingctively, . In addid values of ~ 0.14 μM and ~ 6.5 nM, respectively. Thus, introduction of this single point mutation, A132L, into human PD-1 resulted in 45- and 30-fold enhanced affinities for human PD-L1 and PD-L2, respectively . These two mutants, as well as wild type human PD-1, were expressed in HEK293 cells as soluble Ig fusion proteins linked to the Fc fragment of human IgG1 , and purified from the supernatants of stably transfected cells by affinity chromatography. The purified Ig fusion proteins were examined for binding to PD-L1 and PD-L2 expressed on the surface of HEK293 cells using a flow-cytometry based assay. Consistent with the SPR binding experiments, the human A132L mutant exhibited increased binding to human PD-L1 and PD-L2, compared to the wild type PD-1, while no significant binding was detected between K78A PD-1 Ig and either PD-L1 or PD-L2 . HA PD-1 Ig also showed enhanced binding to mouse PD-L1 and PD-L2 , supporting the feasibility of its use to selectively manipulate the PD-1/PD-Ligand pathways in vitro and in vivo.Identification of the high affinity A132L PD-1 mutant motivated the design of a high-avidity bivalent dimeric PD-1 Ig fusion protein (HA PD-1 Ig). As a negative control, we generated K78A mutant human PD-1 as an Ig fusion, as it does not bind to either PD-L1 or PD-L2 . After treatment with TNF-α to induce a ‘mature’ phenotype, these dendritic cells expressed both PD-L1 and PD-L2  assay as a model for T cell activation. CD4+ T cells purified from the peripheral blood of healthy donors were co-cultured for 5 days with allogeneic monocyte-derived mature dendritic cells pre-treated with the Ig fusion proteins, and subsequent T cell proliferation was measured by 3H-thymidine uptake. HA PD-1 Ig elicited a dose dependent enhanced of T cell proliferation, with observable effects at concentrations as low as 0.5 μg/ml, and a significant increase at 5 μg/ml  model , and RT + IgG. Two groups, RT + IgG and HA PD-1 Ig + RT, received three consecutive fractions of radiation (20 Gy) specifically targeting the tumor, starting on day 1 (about 12 days after tumor cell inoculation), using a hypofractionated image-guided radiation therapy (IGRT)  in the distal inguinal LN (To investigate the L) model  to evaluL) model A. Tumorsy (IGRT)  operatedy (IGRT)   . As expeTo further characterize the anti-tumor effect of our HA PD-1 Ig we compared it to a function blocking anti-PD-L1 monoclonal antibody (mPD-L1 Ab clone 10F.9G2), in combination with radiation therapy using the same 3LL lung tumor model. We found that the synergistic effect of HA PD-1 Ig with radiation therapy in inducing tumor regression was superior to that observed with mPD-L1 Ab, or radiation therapy alone , followed by amputation of the tumor bearing foot (approximately 21 days after tumor inoculation) began exhibiting high numbers of pulmonary metastatic nodules at 3–4 weeks post-treatment. The mean metastatic colony counts were not significantly different between the control, HA PD-1 Ig, and RT + IgG groups . In contrast, the number of metastatic nodules in the RT + HA PD-1 Ig group (1.5 ± 0.58) was significantly reduced, as evidenced by images of the lungs A.5 3LL cells) in the opposite foot and monitored for tumor growth (EM) in both CD4+ and CD8+ T cells in the spleen and draining lymph node (dLN), and by an accumulation of CD8+ T central memory cells (TCM) in the spleen  exhibited close to wild type affinities towards human PD-L2, but significantly reduced affinities towards human PD-L1. These PD-L2 specific mutants could potentially be used to selectively study the role of PD-L1 and PD-L2 in regulating the immune response. A third important class of human PD-1 mutants exhibited increased affinity to both human PD-L1 and PD-L2. In particular, mutation of alanine 132 to leucine, or other hydrophobic residues such as Ile, Phe, Val or Thr, resulted in enhanced binding to both ligands , as observed in cell-surface binding experiments using HEK293 transfected cells.http://exac.broadinstitute.org/ , and iser.ac.uk ). This ser.ac.uk B–C, sugger.ac.uk B–C.While the human and mouse PD-1 mutants tend to show generally similar behavior in terms of ligand binding, including the high-affinity A132 mutants, there are some important differences. In contrast to murine PD-1, where mutations of residues I134 and E136 result in complete loss of binding to both ligands , analogod 0.2 versus 6 μM). A recent study by Cheng et al. reported similar Kd value for the human PD-1/PD-L1 interaction (8.2 μM), but only ~ 4-fold stronger affinity for the PD-1/PD-L2 (2.3 μM) interaction  and 95.1% identity between human and chimpanzee PD-L2 proteins . By comparison, the human and mouse PD-Ligands exhibit ~ 70% identity only. The cross reactivity of the HA PD-1 reagent is a significant feature, which precludes the need to re-engineer the biologic when moving between experimental systems, in contrast to monoclonal antibodies which are typically species-specific.The human high-affinity mutant A132L selected for further studies showed enhanced binding not only to human PD-L1 (45 fold) and PD-L2 (30 fold), but also to the corresponding mouse ligands. Binding of the human A132L mutant to mouse PD-L1 was enhanced 23 fold (Kd ~ 0.23 μM), compared to the endogenous mouse PD-1/PD-L1 (Kd ~ 5.4 μM) interaction, and binding to mouse PD-L2 was increased 96 fold (Kd ~ 29 nM), compared to the endogenous murine PD-1/PD-L2 interaction (Kd ~ 2.8 μM). Due to this binding behavior, the A132L high-affinity human PD-1 (HA PD-1) is suitable for use in 1NPU, (Determination of the human PD-1 (A132L mutant) crystal structure allowed for the detailed analysis of the residues contributing to the ligand-binding interfaces. Human PD-1 exhibits a monomeric IgV domain structure similar to the mouse PD-1 crystal structure . Superp1NPU, . Anotherd in this cell-based system, due to a number of factors , appreciation of the binding responses (The human PD-1 mutant (A132L) exhibiting increased affinity for both PD-L1 (45-fold) and PD-L2 (30-fold) was selected for further studies and the generation of a soluble chimeric Ig fusion protein (HA PD-1 Ig). As expected, compared to wild type PD-1 Ig, the HA PD-1 Ig showed significantly enhanced binding to mature human dendritic cells expressing PD-L1 and PD-L2. Although it is not feasible to define a quantitative Kesponses A–B indicin vitro T cell activation assay, in which the HA PD-1 Ig increased T cell proliferation by at least 50%, . Flow cytometric analysis of tumor-associated  and splenic DCs (not shown) did not show significant increase in surface activation markers after treatment, although there was a trend towards increased expression of PD-L1 , likely induced by IFN-γ release following radiation therapy. Furthermore, the allogeneic MLR studies utilized irradiated DCs, indicating that the enhanced proliferation and cytokine production elicited by HA PD-1 Ig , and TNF receptor Ig fusion proteins (etanercept), each with their specific pharmacological profile.Radiation therapy (RT) induces tumor cell death by eliciting irreparable DNA damage and cell cycle arrest, thus providing a robust source of antigens. By upregulating MHC class I expression, RT enhances antigen presentation and T cell recognition of irradiated tumor cells, sensitizing them to cytotoxic T cell-mediated clearance. RT further affects the tumor microenvironment by inducing increased expression of pro-inflammatory cytokines, such as TNF-α, IFN-γ, IL-1β, IL-6 and IL-12, type-I interferons and chemokines, which stimulate infiltration of immune cells into the tumor . Recent The 3LL cells used in our murine model expresses PD-L1 , and radiation-induced pro-inflammatory cytokines such as IFN-γ have the potential to further upregulate PD-L1 expression in the tumor microenvironment, which could contribute to immune evasion by inhibiting PD-1-expressing effector T cells. Blockade of the PD-L1/PD-1 pathway offers the potential to reverse this immunosuppression, and increase the efficiency of T cell-mediated anti-tumor response.In addition to reducing the size of primary footpad tumors and substantially increasing tumor growth delay, the combination HA PD-1 Ig + RT treatment resulted in a significant reduction of tumor metastasis to the local lymph nodes, as well as distant metastasis to the lungs. The lungs isolated from the combination HA PD-1 Ig and RT group contained very few nodules, compared to all the other groups, which showed extensive metastatic lesions over the entire surface of the lung lobes. Quantification of the metastatic lesions in the lungs showed dramatically reduced metastatic burden in RT + HA PD-1 Ig combination treatment group compared to all the other treatment groups. RT therapy alone slightly reduced the number of metastatic nodules in the lung, but this decrease was not significantly different from the control or HA PD-1 Ig single groups. Interestingly, only the combination radio-immunotherapy resulted in significantly reduced local and distal tumor metastasis in this Lewis lung carcinoma model, consistent with the desired synergistic effect.EM), and CD8 central memory T cells (TCM) in “cured” mice that were re-challenged with a second injection of tumor cells 57 days after the initial tumor challenge. The primary challenge data showed an increase in tumor infiltrating lymphocytes (TILs) at about day 12 in the HA PD-1 Ig + RT combination group , while at later time points no significant influx of TILs was observed (data not shown). Since memory T cells have less stringent requirements for activation than naïve cells e.g., responsiveness to lower concentrations of antigen,  levels at the tumor site. Second, it has been shown in other models that PD-1 blockade reduces the population of highly immunosuppressive myeloid-derived suppressor cells (MDSC), which inhibit anti-tumor immune responses . Third, Intriguingly, administration of HA PD-1 Ig alone did not cause significant difference in tumor growth in this highly invasive, spontaneous metastatic lung tumor model. As indicated tumor immunogenicity is major determinant of responsiveness to immunotherapy. In addition, given the observed variations in the penetrance of autoimmune diseases observed in genetically PD-1 deficient mice, it is likely that host genetic determinants also play an important role. It remains to be determined if monotherapy using HA PD-1 Ig can enhance control of other type of solid tumors, in mice of various genetic backgrounds.One significant advantage of PD-1 mediated immunotherapy, compared to other checkpoint inhibitors, such as CTLA-4, is the reduced rate of undesirable side effects observed in clinical trials . This diAn important feature of the HA PD-1 Ig is that it is fully human, possessing only a single point mutation, as opposed to humanized mouse monoclonal antibodies that have the tendency to elicit the formation of anti-drug antibodies after repeated treatments, which can decrease clinical efficacy and contribute to treatment failure . FurtherIn addition to cancer immunotherapy, HA PD-1 Ig may also find utility for the treatment of infectious diseases. Recent studies have shown that enhancement of T cell activation through PD-1 blockade is beneficial for chronic viral infections with HIV, HCV, HBV and HTLV . In thesin vitro and in vivo (d ~ 0.1 nM), but no appreciable interaction with PD-L2 , and could antagonize the PD-1/PD-L1 interaction  in vivo . Maute ath PD-L2 . This rein vitro and in vivo immunomodulatory properties and make it an attractive potential therapeutic, including bi-specific binding behavior  and extensive cross reactivity with the murine PD-Ligands, which greatly facilitates in vivo characterization. By demonstrating the synergistic effects of combining HA PD-1 Ig treatment with targeted radiation therapy, our findings provide additional strategies for the therapeutic manipulation of the immunosuppressive tumor microenvironment, increased control of primary and metastatic tumor burden and the establishment of immunological memory against tumor recurrence. These results underscore the potential of rational combination therapies as important future modalities for tumor immunotherapy.In summary, using a structure-guided approach, we developed a high-affinity PD-1 Ig fusion protein harboring a single point mutation, which can be used to manipulate the PD-1/PD-Ligand interactions. This bivalent reagent has several features that may contribute to its E. Lazar-Molnar and S.C. Almo have filed a patent on the high-affinity PD-1 Ig. The authors declare no additional competing financial interests.in vitro experiments, analyzed the data and interpreted the results. ES contributed to the in vitro proliferation studies. LS, TQ and IB performed the in vivo tumor experiments and interpreted the results. UAR acquired the crystallography data and provided valuable help with the analysis. EP helped with the protein refolding and purification. SCA, SGN and CG supervised design, data analysis and interpretation. ELM, TQ, LS, CG and SCA wrote the manuscript.ELM designed and performed the


The transfer-free fabrication of the high quality graphene on the metallic nanostructures, which is highly desirable for device applications, remains a challenge. Here, we develop the transfer-free method by direct chemical vapor deposition of the graphene layers on copper (Cu) nanoparticles (NPs) to realize the hybrid nanostructures. The graphene as-grown on the Cu NPs permits full electric contact and strong interactions, which results in a strong localization of the field at the graphene/copper interface. An enhanced intensity of the localized surface plasmon resonances (LSPRs) supported by the hybrid nanostructures can be obtained, which induces a much enhanced fluorescent intensity from the dye coated hybrid nanostructures. Moreover, the graphene sheets covering completely and uniformly on the Cu NPs act as a passivation layer to protect the underlying metal surface from air oxidation. As a result, the stability of the LSPRs for the hybrid nanostructures is much enhanced compared to that of the bare Cu NPs. The transfer-free hybrid nanostructures with enhanced intensity and stability of the LSPRs will enable their much broader applications in photonics and optoelectronics. Graphene as two-dimensional one-atom-thick sheet of carbon has shown its potential for a wide variety of applications1245678910111314161718202122252728293132The graphene/metal hybrid nanostructures are usually fabricated by separate preparation of metal nanostructures and wet etching and transferring the CVD grown graphene onto the metal nanostructures5343537Here, we develop an etching and transfer-free technique for one-step fabrication of the graphene/Cu NPs hybrid nanostructures by direct growth of graphene on the hemispherical copper nanoparticles to form graphene-coated hemispheres with Cu cores. The as-grown graphene/Cu NPs hybrid nanostructures exhibit enhancements in both intensity and stability of the LSPRs due to the graphene-induced enhancement in the light–matter interactions and the surface passivation. The as-grown graphene on the Cu NPs is beneficial to their full electric contact and strong interactions on account of the larger adhesion energy. 10-fold enhancement of fluorescence from the dye coated hybrid nanostructures has been obtained due to graphene-induced enhanced LSPRs, which has been supported by finite-difference time domain (FDTD) calculation. Moreover, the stability of the LSPRs for the hybrid nanostructures is much enhanced compared to that of the bare Cu NPs due to the surface passivation of the graphene sheets covering completely and uniformly on the Cu NPs. Our findings might open up a new avenue to realize high quality graphene/Cu NPs hybrid nanostructures and fulfil their practical applications in photonics and optoelectronics.−1, the G peak at 1580 cm−1 and the 2D peak at 2659 cm−1 are observed, respectively, which confirms the direct synthesis of few layer graphene on Cu NPs. In particular, the D peak at 1337 cm−1 originates in the lattice disorder probably arising from the etch process for analysis or the high curvature of Cu NPs surfaces, rather than the practical defects in the graphitic lattice.The specific fabrication procedures of the as-grown graphene on the hemispherical copper nanoparticles are schematically summarized in 29To shed light on the specific function of graphene coupling to the LSPRs of the metal nanoparticles, the steady-state optical absorption spectra for the Cu NPs with or without the graphene coating are compared and shown in 42In-house-generated finite-difference time-domain (FDTD) simulations are performed to further investigate the LSPRs enhancement induced by the graphene coating. We calculate the reflection spectra of the graphene-coated Cu NPs arrays versus different numbers of the graphene layers as shown in 2 exhibits its maximum at the surface of Cu NPs and shows exponential decay along the direction perpendicular to it, demonstrating the excitation of the LSPRs. In this case, the presence of graphene sheets leads to a strong localization of the field at the graphene–copper interface, as shown in 2O), cupric oxide (CuO) and copper hydroxide (Cu(OH)2)192023The surface of the bare copper is prone to proceed oxidation as exposed to the ambient air, leading to the development of layers consisting of cuprous oxide  initial formation of cuprous oxide (Cu2O), (c) formation of cupric oxide (CuO) islands, and (d) further formation of copper hydroxide (Cu(OH)2)3/2 and Cu 2p1/2 at binding energies of 932.2 eV, 952.3 eV  and 932.8 eV, 952.5 eV , respectively, which indicates negligible oxidation of the graphene/Cu NPs hybrid nanostructures after thermal treatment. However, in case of the bare Cu NPs (2O (932.7 and 952.3 eV), CuO (933.7 and 953.4 eV), and Cu(OH)2 (935.1 and 954.5 eV) are formed after thermal treatment for 4 hours. The photograph images of both thermal treatments are shown in To further evaluate the graphene passivation, X-ray photoelectron spectroscopy (XPS) measurement is carried out to analyze the metal composition. e Cu NPs , right, In conclusion, we have demonstrated a novel and facile route to fabricate the hybrid nanostructures of as-grown etching-free graphene on Cu NPs with full electric contact and strong interactions by the chemical vapor deposition (CVD) process. Few-layer graphene can be directly synthesized on the Cu NPs, which is verified from the Raman spectroscopy and HRTEM micrographs. The intensity of the LSPRs supported by the graphene/Cu NPs hybrid nanostructures has been improved, and results in an 10-fold enhanced fluorescent intensity from the dye coated hybrid nanostructures. The stability of the LSPRs for the hybrid nanostructures has been much enhanced compared to that of the bare Cu NPs due to the passivation of the graphene coating. The transfer-free hybrid nanostructures with enhanced intensity and stability of the LSPRs might play a significantly important role in the development of Cu-based plasmonic nanostructures, hybrid nanophotonic and optoelectronic devices and ultra-large-scale integrated circuits.−4 Pa. Thermal annealing then preformed in the low pressure chemical vapor deposition (LPCVD) system at the atmosphere of hydrogen (H2) and argon (Ar) gas to obtain copper nanoparticles with morphology-controllable characteristics. After the introduction of methane (CH4) gas into the chamber at the proper temperature of 1000 °C, graphene/copper nanoparticles hybrid nanostructures can be realized directly after the rapid cooling without time-consuming transfer method. The gas flow rates of CH4, H2 and Ar were 7, 30, 200 standard cubic centimeters per minute (sccm), respectively. The ramping rate of the temperature was approximately 40 °C/min, followed by rapid cooling to room temperature (25 °C) with protective gas configuration of H2 and Ar.Fabrication and characterization of graphene/Cu NPs hybrid nanostructures: The copper films of 40 nm were fabricated on the pre-cleaned quartz substrates  via thermal evaporation with pieces of copper foil  at a base pressure of 5 × 103 for 10 h and rinsed in deionized water for 20 min subsequently to etch the Cu cores prior to the micro-Raman spectroscopy analysis. Absorption spectra were measured using UV-vis spectrophotometer . The chemical composition of the specimens was characterized by X-ray photoelectron spectroscopy .Scanning electron microscopy  and high resolution transmission electron microscopy  were utilized to measure the surface morphologies of the samples. Raman spectra  were obtained with a 532 nm laser to analyze the quality of graphene. In order to remove the influence of Cu fluorescence peaks overlapping with typical graphene peaks, the specimens were thoroughly soaked in 20% HNO−4 Pa. The fluorescence spectra were obtained at the excitation light of 480 nm from fluorescence spectrophotometer .A 25 nm thick emitting layer of 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) was thermal evaporated on the specimens of graphene films, bare Cu NPs, graphene/Cu NPs and quartz substrates at a base pressure of 5 × 10How to cite this article: Li, Y.-F. et al. As-grown graphene/copper nanoparticles hybrid nanostructures for enhanced intensity and stability of surface plasmon resonance. Sci. Rep.6, 37190; doi: 10.1038/srep37190 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Since the successful exfoliation of graphene, various methodologies have been developed to identify the number of layers of exfoliated graphene. The optical contrast, Raman G-peak intensity, and 2D-peak line-shape are currently widely used as the first level of inspection for graphene samples. Although the combination analysis of G- and 2D-peaks is powerful for exfoliated graphene samples, its use is limited in chemical vapor deposition (CVD)-grown graphene because CVD-grown graphene consists of various domains with randomly rotated crystallographic axes between layers, which makes the G- and 2D-peaks analysis difficult for use in number identification. We report herein that the Raman Si-peak intensity can be a universal measure for the number identification of multilayered graphene. We synthesized a few-layered graphene via the CVD method and performed Raman spectroscopy. Moreover, we measured the Si-peak intensities from various individual graphene domains and correlated them with the corresponding layer numbers. We then compared the normalized Si-peak intensity of the CVD-grown multilayer graphene with the exfoliated multilayer graphene as a reference and successfully identified the layer number of the CVD-grown graphene. We believe that this Si-peak analysis can be further applied to various 2-dimensional (2D) materials prepared by both exfoliation and chemical growth. Single-layer graphene is a promising material for nanoelectronics because of its high electrical transport properties that can be controlled by an applied electric field2. The high optical transparency3, chemical stability4, and high mechanical strength5 render single-layer graphene versatile for nanophotonic and optoelectronic applications and flexible electronics. The importance of multilayer graphene (MLG) has also rapidly grown for the past few years because of its unique functionality and wide applicability in various optical and electrical nanodevices. Gate-tunable bandgaps particularly enable the fundamental investigation of graphene-based optoelectronics8. In addition, multilayer graphene offers an unprecedented opportunity to flexible electronics, thereby providing optically transparent, atomically thin, and ultra-flexible electrical contacts  van der Waals heterostructures (vdWs))9. The most remarkable feature of these layered 2D materials including graphene is the layer-number-dependence of the physical properties even in the same material. Therefore, the precise control of the number of layers in 2D materials has become one of the most critical steps in fabricating 2D nanodevices. In this regard, controlling the layer numbers of 2D van der Waals materials with an unambiguous and reliable identification method is required first.Graphene has attracted great interest from researchers in various fields since the first successful separation of monolayer graphene from graphite2 thickness11. Other optical techniques have been introduced to identify the number of layers of multilayer graphene, including Raman spectroscopy17 and Rayleigh scattering18. Raman spectroscopy is more widely used because it not only provides more information about the layer number of a given sample, but also reveals important physical properties, such as phonons, electron–phonon coupling, band structures, and interlayer coupling of multilayer graphene. The most discernable two peaks in the Raman spectrum of graphene and graphite are the G-peak (1580 cm−1) and the 2D-peak (~2700 cm−1). For exfoliated graphene the number of layers can be reliably determined from the line shape of the 2D peak for fewer than 7 or 8 layers14. The intensity ratios of G/2D15 and G/Si16 are also introduced to facilitate the counting process using the Raman G- or 2D-peak. However, they are reliable only in limited ranges. For example, the G-peak intensity of a thin-layer region tends to increase with the function of the layer number, but decrease in the thick layer region (>18 layers)17. Moreover, the 2D-peak of the multilayer graphene is composed of multiple peaks. Therefore, the countable number is limited for thin layers. Xiao-Li et al. recently reported the layer number identification of exfoliated multilayer graphene with a wide countable range (1–100 layers) using the Si substrate peak intensity17. They used multiple reflection interference method of constitution of air, multilayer graphene, SiO2, Si with complex refractive index and the thickness of each medium. No reversible point limits the identification range in the case of the Si-peak intensity. Moreover, 532 nm excitation gives a good optional choice for layer number determination for various SiO2 layer thicknesses  graphene. For CVD-graphene, since the 2D-peak line-shape changes significantly by the twisted angles between the layers even on the same layer numbers, the number identification becomes quite complicated than the exfoliated graphene. The verifying number of randomly stacked large-scale multilayer graphene accompanied with a twisted angle is an essential step for transparent and flexible electronic applications. Hence, a credible avenue capable of identifying the number of graphene is demanded.The optical contrast technique is a rapid and easy method of confirming the layer number of multilayer graphene representing the sample thickness by the contrast of reflection spectra or color difference. However, the technique is only useful in relative thickness comparison, and has a limited range that can be measured depending on the SiO25. Only the Si-peak exhibited a gradual intensity variation as the number of layers increased or decreased. In addition, the peak positions remained unchanged for all of the graphene layers. This comparison confirmed that the Si-peak analysis was robust, and a powerful characterization tool for identifying the number of layers in CVD-grown multilayer graphene.In this work, we identified the usefulness of the Si-peak analysis in determining the number of layers of CVD-grown graphene using 532 nm excitation. We compared the optical characteristics between an optical image and the Raman images of the Si-, G-, and 2D-peaks to determine the number of layers of graphene grown by CVD. We confirmed that the CVD-grown graphene has many disorders because of the twisted angle between the layers, which caused 2D- and G-band variations even at the same layer number25. Consequently, without knowing the exact layer number and layer boundaries, further material characterizations and analyses of CVD-grown multilayered graphene are not possible. However, the Si-peak intensity mapping image  with respect to that of the SiO2/Si substrate [I(Si0)]. Then we co-plotted I(Si) and the corresponding peak positions as a function of the number of graphene layers [Fig. −1) and the linewidth (FWHM ~19.1 ± 0.3 cm−1) of the Raman spectra of the Si-peak remained unchanged. All these tendencies agree well with the observations from the CVD-grown multilayered graphene sample in Fig. I(G)] with respect to I(Si0) and co-plotted them with the corresponding peak positions as a function of the number of graphene layers  were normalized with the Si-peak intensity of the SiO2/Si substrate [I(Si0)].In Fig. Figure I(Si)/I(Si0) from both the exfoliated (thick brown line) and CVD multilayer graphene (black squares). By directly comparing these values, the layer numbers of the thick multilayer CVD graphene  were successfully determined. We note that there are several advantages in using this Si-peak for identifying the layer number in CVD graphene. First, the described method is simple and only requires Si-peak intensity data. In addition to the layer number identification, the Raman mapping results can be used to obtain the local information of the D-, G-, and 2D-peaks for further analysis, such as structural disorder and electrical doping. Second, the described quantity of normalized I(Si) [I(Si)/I(Si0)] can be universally applied to identify the number of layers for both exfoliated and CVD graphene, which reveals that the Si-peak is not significantly affected by undesirable experimental factors such as PMMA residues and structural disorders commonly observed in CVD-grown multilayer graphene. Third, the Si-peak shows no angle dependency in twisted multilayer graphene, which is a key limitation of other Raman spectroscopic methods.Finally, we used the normalized Si-peak intensity data measured from the exfoliated multilayer graphene Fig.  as a refWe demonstrated a reliable identification of the number of layers of CVD-grown multilayer graphene. We obtained the normalized Si-peak intensities of exfoliated multilayer graphene, and applied it to CVD-grown multilayer graphene. CVD-grown multilayer graphene has complex G- and 2D-band variations depending on the crystal disorder even in the same number of layers. However, using normalized Si-peak intensities to identify the number of graphene layers was useful for both CVD-grown and exfoliated multilayer graphene. We believe that the Si-peak analysis is the most powerful tool for determining the number of layers of multilayer graphene samples although the further study for different types of substrates would be necessary. Furthermore, this identification method can be exploited for Van der Waals heterostructures made of various 2D materials such as hBN and TMDCs, when substrate related peaks are found and the relationship with the number of layers is verified.27. The thick multilayer graphene was synthesized using a 300 nm Ni metal catalyst, and the thin multilayer was grown on a Cu(400 nm)/Ni(300 nm) metal catalyst. The metal catalyst films were deposited onto a thermal oxidized 300 nm thick SiO2/Si substrate, which was then heated up to 1,000 °C inside a CVD under H2 atmosphere, and the graphene was then grown with flowing gas mixtures of H2:CH4 = 10:5 (sccm) for 20 min. After synthesizing the graphene, the polymethyl methacrylate (PMMA) was coated on the graphene at 3000 RPM for 30 s. The PMMA/graphene/metal catalyst was separated from the Si substrate during floating on a buffered HF (BOE) for several minutes. The metal catalyst was then etched by floating on a 0.1 M ammonium persulfate solution. After rinsing the PMMA/graphene with DI-water several times, the PMMA/graphene was transferred onto the target substrate and baked at 180 °C for 30 minutes, increasing the adhesion between the graphene and target substrate. The PMMA was removed with acetone and IPA.The multilayer graphene samples were synthesized using a Cu/Ni metal catalyst by chemical vapor deposition2 substrate.The Raman mapping images were obtained using a NTEGRA Spectra from NT-MDT, equipped with a thermoelectric (TE) cooled CCD and a ×50 objective lens (NA = 0.75). The excitation laser with a wavelength of 532 nm from a diode laser was used for all Raman measurements. The Si-, G-, and 2D-peak intensities were extracted from the full-spectrum mapping results and compared after normalization with the Si-peak intensity on the SiOSupplementary Information


In a number of animal species, the growth-associated protein (GAP), GAP-43 , has been implicated in the regulation of presynaptic vesicular function and axonal growth and plasticity via its own biochemical properties and interactions with a number of other presynaptic proteins. Changes in the expression of GAP-43 mRNA or distribution of the protein coincide with axonal outgrowth as a consequence of neuronal damage and presynaptic rearrangement that would occur following instances of elevated patterned neural activity including memory formation and development. While functional enhancement in GAP-43 mRNA and/or protein activity has historically been hypothesized as a central mediator of axonal neuroplastic and regenerative responses in the central nervous system, it does not appear to be the crucial substrate sufficient for driving these responses. This review explores the historical discovery of GAP-43 (and associated monikers), its transcriptional, post-transcriptional and post-translational regulation and current understanding of protein interactions and regulation with respect to its role in axonal function. While GAP-43 itself appears to have moved from a pivotal to a supporting factor, there is no doubt that investigations into its functions have provided a clearer understanding of the biochemical underpinnings of axonal plasticity. The growth-associated protein (GAP), GAP-43, has been known under various designations (see below) over the span of 40+ years in the course of separate yet overlapping investigations into phosphoproteins that contribute to plasticity and growth of the presynaptic terminal. Exploring this history has revealed the basic properties of this growth- and plasticity-related protein that set the foundation for work to determine whether this protein was a choke point for axonal malleability during states of plasticity or a component of a larger presynaptic proteome. From this historical perspective, it is clear that this presynaptic phosphoprotein of 43 kDa generated some excitement as it rose to the forefront of other polypeptides that were also under investigation. However, it also became clear that this protein functioned as a cog in a larger set of proteins and kinases to coordinate changes in axonal structure and function during times of plasticity.35S] methionine suggesting a slow transport rate of between 2 mm/day and 4 mm/day for these proteins  following intraocular injection of [Skene and Willard  detectedin vitro, Zwiers et al. (While studying the effects of adrenocorticotropic hormone (ACTH) on the phosphorylation of synaptic plasma membrane proteins s et al.  found ths et al. . In thiss et al.  was desis et al. . The phos et al. , 1979. As et al. ,b that es et al. . Proteins et al. ,b in a cs et al. . Severals et al.  and withs et al. . These fin vivo state of the enzymes at the time of tissue assessment  rose sharply peaking at 134 PubMed reports in 1995 but showing a decreasing trend over the past 20 years (lowest in 2016 at 65 publications). It appears that this protein was initially hailed as playing a pivotal role in axonal outgrowth but has since become a contributing factor as part of a larger group of axonal growth-permissive proteins. The study of GAP-43 has revealed a tight relationship between its expression and axonal structural plasticity during development, a rise in its expression levels following axonal injury during the regeneration phase and an association with input-dependent neuroplastic processes, such as LTP and memory formation, in conjunction with reorganization of axonal terminal fields Denny, . The funExpression of GAP-43 mRNA is restricted to the nervous system, and in particular, axons, where levels increase during periods of neurite outgrowth  imparts a marginal effect on GAP-43 phosphorylation  and can activate presynaptic PKC to phosphorylate various substrates such as GAP-43 to regulate cytoskeletal dynamics  because low frequency, nonpotentiating stimulation did not increase (or decrease) GAP-43 phosphorylation , behavioral recovery as assessed with a pellet-reaching task occurred during a 14-day period after MCAo and in the affected cervical gray matter, GAP-43 staining on corticospinal tract axons was significantly increased during the same period while synaptophysin increased in axonal terminals at 28 days , inhibiting Nogo-A expression with an anti-Nogo-A monoclonal antibody , the transcriptional and post-transcriptional regulation of the mRNA and the post-translational modification (phosphorylation) and protein localization were intensely scrutinized as a means to facilitate central nervous system plasticity. Many growth permissive events in the central nervous system are associated with the augmentation of GAP-43 levels suggesting it may have been a critical factor for plasticity-associated axonal function. While upregulation of GAP-43 protein phosphorylation status, enhanced stability of GAP-43 mRNA and inhibition of Nogo-A/Nogo66 inhibitory processes have all been shown to promote axonal outgrowth, GAP-43 appears to play a more permissive role in axonal plasticity being an important node in coordinating various other critical factors. If nothing else, the exploration of GAP-43 function and upstream and downstream regulators has stimulated the search for targeted therapeutics directed to facilitate CNS regeneration after axonal injury (see Ribas and Costa, MRH wrote and edited the review manuscript and is responsible for all content.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Various supercell models are constructed in order to reduce the strain induced by the lattice mismatch. The results show that stanene interacts overall weakly with graphene via van der Waals (vdW) interactions. Multiple phases of different crystalline orientation of stanene and graphene could coexist at room temperature. Moreover, interlayer interactions in stanene and graphene heterostructure can induce tunable band gaps at stanene’s Dirac point, and weak p-type and n-type doping of stanene and graphene, respectively, generating a small amount of electron transfer from stanene to graphene. Interestingly, for model  The res2Te3 (111) substrate by molecular beam epitaxy (MBE). Xu et al. [Most recently, a new 2D material, stanene (the form of 2D stannum), firstly proposed by Liu et al. , has beeu et al.  found thTechnically, it is possible to fabricate a heterostructure of stanene on a suitable substrate, in order to form honeycomb-like bilayer atomic structure. Stanene has a hexagonal lattice, as well as the requirement of lattice status of the substrate. The lattice mismatch between the substrate and the stanene should be small, and it should be energetically favorable to stanene to grow in a quasi-two-dimensional growth mode. As one of the popular 2D materials, we propose a question whether stanene can grow on a graphene substrate or stanene/graphene (Sn/G) can form a 2D heterostructure with promising structural and electronic properties.In this work, we design a new 2D stanene/graphene heterostructure and study its geometric and electronic properties by using first-principles calculations. The results show that stanene interacts overall weakly with graphene via vdW interactions. Therefore, their intrinsic electronic properties can be preserved in stanene/graphene heterostructure. Moreover, interlayer interactions in stanene/graphene heterostructure can induce tunable band gaps at stanene’s Dirac point, and weak p-type and n-type doping of stanene and graphene, respectively. Our paper is organized as follows. In the “−5 eV. A vacuum layer of at least 20 Å is used.Our theoretical calculations are performed in the framework of density functional theory (DFT)  as impleFor the monolayer graphene and free-standing low-buckled stanene, the lattice constants we obtained from LDA are 2.45 and 4.56 Å, respectively, which agree well with the reported values of 2.46 and 4.67 Å for graphene and stanene, respectively , 54. Notα equal to 9. This configuration will be represented as The heterostructures along with their structural parameters are listed in Table Figure LSn/G in Table LSn and LG are the lattice constants for the particular unrelaxed supercells of stanene and graphene, respectively. The lattice parameter of Sn/G heterostructure is fixed to be (LSn + LG)/2 with a small lattice mismatch for both stanene and graphene. After fully relaxation, it is found that LSn/G is very close to LG, which indicates that there is almost no strain in graphene layer. The lattice mismatch between the periodic unrelaxed supercell of monolayer stanene and graphene is defined as mismatch = |LSn − LG|/LG, and it is a quite small value as shown in Table a and a0 are the relaxed (bilayer) and unrelaxed primitive lattice constants.where θ and buckling height Δ in stanene will be slightly affected by the strain as shown in Table θ is uniform, as shown as red circle in Fig. θ shown in Table θ to be slightly smaller and the buckling height Δ to be higher. There are opposite results when the strain > 0, the stanene layer is forced by a tensile strain. The change of electronic properties caused by the variation of bond angles will be discussed below.As shown in Table Eb, per Sn atom) in the Sn/G bilayer asThe vdW interaction between the layers requires special attention. To quantitatively characterize the interlayer interaction strength, we define a binding energy  between stanene and graphene has been fully relaxed, getting up to an identical distance (3.4–3.5 Å) and a uniform buckling height Δ = 0.8 Å as shown in Fig. where mentclass2pt{minimmathrm{G} $$\end{dasis set . It indistance 3.–3.5 Å anThe energetics of the stanene overlayer can be addressed by examining the energy per Sn atom defined as:μSn is the chemical potential set to the energy per atom of bulk Sn. The calculated energies per Sn atom using different heterostructures in Table Ec is positive, indicating that the 2D structure is higher in energy than the 3D diamond structure. Among all the heterostructures we have considered, where E(k) = ± ℏνF|k| around the Fermi levels [Two-dimensional honeycomb structures exhibit a symmetry between the sublattices and therefore have a linear energy dispersion in the vicinity of the Dirac points at K point. Figure i levels , the chai levels , 57:4\doΔED is the shift of graphene’s Dirac point (ED) relative to the Fermi level (EF), that is ΔED = ED − EF. Our calculated charge carrier concentrations are Nh (Sn) = 1.4 × 1012 cm−2 and Ne (G) = 1.6 × 1011 cm−2 for stanene and graphene in bilayer, respectively. These values are larger than the intrinsic charge carrier concentration of graphene at room temperature ( n = πkB2T2/6ℏνF2 = 6 × 1010cm− 2) [where 10cm− 2) . Further10cm− 2) . The selFigure eft\sqrt{}\right/\When focusing on Fig. nderbilt , in whicnderbilt  (WCCs) dIn conclusion, by first-principle calculations, we found it is possible to synthesize stanene on the graphene substrate without destroying its characteristics of the Dirac-fermion-like linear dispersion around Dirac points, due to the weak van der Waals interlayer interaction. In addition, multiple phases of different crystalline orientation of stanene and graphene could coexist at room temperature based on our energetics analysis. Moreover, interlayer interactions in stanene and graphene heterostructure can induce tunable band gaps at stanene’s Dirac point, and weak p-type and n-type doping of stanene and graphene, respectively, generating a small amount of electron transfer from stanene to graphene. For stanene on graphene, the gap created by the substrate effect is of the same order as that induced by the SOC effect. Interestingly, for model


We explore the contributions to the electrical resistance of monolayer and bilayer graphene, revealing transitions between different regimes of charge carrier scattering. In monolayer graphene at low densities, a nonmonotonic variation of the resistance is observed as a function of temperature. Such behaviour is consistent with the influence of scattering from screened Coulomb impurities. At higher densities, the resistance instead varies in a manner consistent with the influence of scattering from acoustic and optical phonons. The crossover from phonon-, to charged-impurity, limited conduction occurs once the concentration of gate-induced carriers is reduced below that of the residual carriers. In bilayer graphene, the resistance exhibits a monotonic decrease with increasing temperature for all densities, with the importance of short-range impurity scattering resulting in a “universal” density-independent  conductivity at high densities. At lower densities, the conductivity deviates from this universal curve, pointing to the importance of thermal activation of carriers out of charge puddles. These various assignments, in both systems, are made possible by an approach of “differential-conductance mapping”, which allows us to suppress quantum corrections to reveal the underlying mechanisms governing the resistivity. The reason for this may be traced to the ultrathin structure of this material, which allows its conduction to be influenced by a variety of different scattering mechanisms23. Phonon-mediated scattering, for example, can be generated by graphene’s intrinsic modes9, but can also be due to rippling11 or the underlying substrate13. Impurity scattering, on the other hand, is usually attributed to the presence of long-range charged impurities in the dielectric substrate19, and of short-range neutral defects in the graphene itself22. Collectively, these mechanisms may combine to yield complicated variations of the resistance as a function of both temperature (T) and carrier density . A problematic task in experiment is that of separating out the different contributions, a job that is made all the more challenging at low temperatures by the emergence of quantum corrections29. Most notable of these are those arising from weak (anti) localization27 and electron-electron scattering29, although Kondo physics has also been invoked to account for the behaviour observed in this regime34.In spite of the interest that graphene has attracted over the past decadeµ). The situation in graphene is more complicated, however, not only for the reasons identified above. Electrons and holes reside on a two-dimensional Dirac cone, placing significant phase-space restrictions on electron-phonon scattering37. The large (>160 meV) energy of the graphene optical modes is additionally understood to suppress their importance, and the overall message is that phonon scattering is weak37. Room temperature mobility as large as ∼20,000 cm2/Vs is therefore expected for graphene on SiO219, while experiment has demonstrated values an order of magnitude larger for suspended layers6. In many other experiments (performed with graphene on SiO2), however, observed mobility values may be smaller than 1000 cm2/Vs39, suggesting that the contribution to the resistivity from impurity scattering can be much more important than in conventional semiconductors.In conventional semiconductors near room temperature, and with modest levels of doping, phonon scattering is typically the dominant process limiting carrier mobility (2 transistors over a wide range of temperature (3–300 K), and for densities spanning the electron and hole branches of the Dirac cone. Due to their different energy dispersions, the importance of Coulomb and short-range impurities is expected to be very different in monolayer and bilayer graphene22. We therefore perform a comparative study of transistors fabricated with these materials. While prior work has largely focused on the resistance variations exhibited by graphene under linear transport23, far fewer studies44 have applied differential-conductance measurements to this problem. Here we use measurements of differential-conductance, performed as a function of temperature, density and drain bias, to identify the different contributions to graphene’s resistance. Crucially, this allows us to identify resistivity contributions, free of the influence of quantum corrections. Our findings in both the monolayer and bilayer systems are in good agreement with the theories45 proposed by Das Sarma and his colleagues.In this article, we study the resistance of graphene/SiO23. To make a direct connection to these earlier works, in Fig. Vg) and temperature. In Fig. 22.We begin our discussion by first of all addressing the temperature dependence of graphene’s linear transport, a problem that has previously been widely investigatedn or p). While these variations might appear to suggest a common origin, in both materials, this, in fact, is not the case. Rather, the observed behaviour is completely consistent with what one expects when scattering in the monolayer is dominated by screened Coulomb impurities22, and that in the bilayer is governed by unscreened short-range disorder22.As a final comment, we note that Fig. gd) as a function of these parameters.  Fig. gd−1 as a function of Vd and Vg, the right-hand panels plot the differential resistance at fixed density (see figure caption) and as a function of temperature. The Dirac point is identified as a pronounced peak at Vg = 14 and Vg = −5 V, for monolayer and bilayer graphene, respectively, as indicated by the dotted lines in the left-hand panels. The main features of these contours are: (1) The zero-bias peak in differential resistance that is strongly suppressed with increasing temperature, washing out around 40–50 K; (2) The pronounced temperature-dependent variations of gd−1, away from the zero-bias peak, which appear to be of opposite sign for the monolayer and bilayer systems.While linear transport provides a useful tool to reveal the role of different scattering mechanisms, for a more complete picture, offering energy resolution, it is necessary to make use of nonequilibrium studies. It is this approach that we apply here, where we perform differential-conductance mapping of graphene, as a function of drain bias, gate voltage, and temperature. The value of this approach is highlighted in Fig. gd−1) is consistent with the influence of quantum corrections, most notably weak localization and electron interactions46. As can be seen in the data of Fig. 46. Furthermore, in the upper inset to Fig. 28, the amplitude of the zero bias peak is only reduced by ∼50% compared to its size at zero magnetic field. This indicates that this feature arises from a combination of weak localization and electron interactions, both of which become increasingly important as the temperature is lowered and carrier coherence is enhanced46  but the resistance no longer drops at low temperatures, instead showing a tendency to saturation. This is presumably due to self-heating of the graphene carriers under the strongly nonequilibrium voltage47; the voltage of 20 mV, for example, corresponds to an equivalent temperature of 200 K, consistent with the temperature below which the resistance saturates in Fig. Having described how the quantum corrections may be suppressed in our studies, in this section we focus in more detail on the resistance variations exhibited by the monolayer Fig.  and bilang = 1.7 × 1012 cm−2. With the quantum corrections suppressed by the application of the DC bias, the resistance at higher temperatures is characterized by a monotonically-increasing  variation. Similar behaviour is apparent, also, in the lower inset to Fig. 12 cm−2.In Fig. While we have focused thus far on the behaviour exhibited by monolayer graphene, in panels (a) & (b) of Fig. T contribution over the range of interest here7. With regards to scattering from optical phonons, the large energies associated with these modes is often thought to render them unimportant at or below room temperature. However, recent first-principles calculations9 based on density-functional theory suggest that, above ∼200 K, optical-phonon scattering may actually become stronger than that due to the acoustic modes, resulting in a metallic resistivity that shows a more rapid increase than linear-in-T.Before discussing the implications of our experimental results it will be helpful to first of all summarize some of the theoretical predictions regarding the sources of resistivity in graphene. Phonons should always give rise to an increasing  resistivity with increasing temperature, with acoustic phonons providing a linear-in-2 substrate and to neutral defects in the graphene layer. With regards to charged impurities, the influence of these is strongly dependent on the screening generated by carriers in the graphene. Screening enters the matrix element for the scattering process via the dielectric function, which in turn depends upon the polarizability of the material. The key point here is that the dependence of this function on temperature and scattering wavevector is very different in monolayer and bilayer graphene, resulting in very distinct screening properties in these materials22. Most notably, in monolayer graphene the polarizability shows a nonmonotonic dependence on temperature, which should give rise to a similarly nonmonotonic variation of the resistivity. Hwang and Das Sarma20 have calculated this behaviour analytically, accounting for the influence of thermal carriers via usual Fermi-Dirac statistics, obtaining asymptotic expressions for the monolayer conductivity:σ0 = σ(T = 0), TF is the Fermi temperature, rs is the Wigner-Seitz radius and:rs = 0.88 (for graphene on SiO2), equation . A critical difference with the monolayer, however, is that scattering from short-range impurities is expected to be much more important in bilayer material. The short-range scatterers give rise to a strong insulating temperature dependence of the resistivity in this system, which can overwhelm any signature from charged impurities.  While the resistivity correction arising from short-range scatterers can only be evaluated numerically, the authors of ref. σ(T/TF)/σ0) that is independent of density.Das Sarma 45 by the Maryland group, the authors addressed explicitly the thermal activation of carriers out of localized puddles arising from the disorder background. Here the authors discussed the puddles as giving rise to a “two-component” model of transport; the first involving usual diffusive transport in the graphene layer, while the second involves the thermal activation of carriers out of the puddles. The activated transport is predicted45 to give rise to an insulating conductivity near the Dirac point in both monolayer and bilayer graphene systems (equations (kBT < s):s is the strength of the potential fluctuations in graphene and is typically expected to be in the range of 10–100 meV. The thermal activation is expected to be most significant in the bilayer system, in which the extent of puddle formation should be much stronger, leading to larger characteristic values of s45. Nonetheless, evidence for the activation has been found in disordered, CVD-grown, monolayer graphene48. Here, the competition between thermal activation, temperature-dependent screening, and phonon scattering was found to result in a nonmonotonic variation of the conductivity, in the crossover regime between insulating and metallic states.In later workquations , respect2 substrate, there is evidence to suggest that it can be observed at sufficiently low densities in clean, suspended material53.The presence of puddling in typical experiments usually obscures the intrinsic variations of the conductivity that are expected for graphene. Here, the conductivity near its Dirac point is predicted to follow the semiclassical transport theory proposed in ref. Vd = 10 mV) applied to suppress its quantum-corrections. In the inset to Fig. Vg ≤ 14 V). These data are re-plotted in the main panel of the figure, by rescaling them relative to their low-temperature resistance (at 3 K). Starting from a gate voltage of −10 V, the Fermi level lies well within the valence band , however, the resistance develops a nonmonotonic character, the onset of which appears at progressively lower temperatures for larger gate voltages. For Vg = 6 V, for example, the crossover to an insulating resistance occurs around 250–300 K; by the time the Dirac point is reached, however, the onset has shifted down to ∼150 K.With this understanding, we now consider the implications of our experimental results in the light of the predictions above. We begin, in Fig. see Fig.  and the ng) that they imply. From this table, we see that the data in this figure span a wide range of carrier density, from ng ∼ 2 × 1012 cm−2 in the highest case to almost ng ∼ 0 near the Dirac point (at Vg = 14 V). In Fig. 11 cm−2), the gate-induced carrier density is larger than that due to both residual impurities (n*) and thermal carriers (nth), even at room temperature. . According to Park et al., the phonon-limited resistivity is governed by acoustic modes at low temperatures, whereas at temperatures above ∼150 K the role of optical phonons becomes more important. It is this crossover in scattering that we attribute to the rapid upturn of the resistivity seen in experiment beyond 150 K. From this analysis we therefore conclude that, when the concentration is dominated by the influence of gate-induced carriers, the resistivity of graphene is essentially determined by phonon scattering. This situation changes when the gate-induced concentration of carriers decreases, however, as we now discuss.The data of Fig. ng = 5 × 1011 cm−2 and it is clear that the scaled resistance deviates from a purely metallic variation that is the signature of electron-phonon scattering and develops, instead, a nonmonotonic dependence on temperature. The onset of this behaviour initially occurs near room temperature, but decreases to ∼150 K as ng → 0. A clue as to the origins of this nonmonotonicity, is provided again in Section ng < 5 × 1011 cm−2, one enters a new regime where the full carrier concentration is strongly modified by the contribution from charged-impurity induced residual carriers. On the basis of these observations, we attribute the nonmonotonic variations seen in Fig. 20. To establish this point, in Fig. T << TF), where the prediction of equation . It is clear that good agreement with this line is obtained for the lowest density studied, where the gate-induced concentration (ng = 1.4 × 1011 cm−2) is in fact smaller than that (∼2 × 1011 cm−2) due to the Coulomb impurities. As the gate voltage is used to produce heavier hole doping, however, the conductance rises rapidly above the dotted line in Fig. ng < 5 × 1011 cm−2), the resistivity data closely follow the universal variation expected20 for scattering from screened Coulomb impurities, indicating that this mechanism dominates over phonon scattering in this limit. As the density is increased, however, the data in Fig. ng > 1012 cm−2).As the gate voltage is increased beyond 6 V in Fig. equation  is expece2/h per square /σ0). This behaviour can be clearly seen in the main panel of Fig. T/TF for a number of electron densities in the range of ng = 1.2 – 2.9 × 1012 cm−2 (see the table included in Section Vg to ng for the bilayer). Over this density range, where the contribution from gate-induced carriers dominates the total electron or hole density  than those in the main panel. Here it is clear that the data progressively deviate from the density-independent curve, as ng is reduced to 1.0 × 1011 cm−2. Since this concentration corresponds to the situation where ng < n*, one might expect that the transport should be increasingly limited by puddling behaviour associated with charged background impurities. Consistent with this, in this regime, we find that the temperature dependence of the conductance is well described by the prediction of equation  for the potential-fluctuation strength.Turning to the variations exhibited by bilayer graphene, these are somewhat easier to analyse since they show no evidence of any metallic variation that would provide a signature of electron-phonon scattering. The first point we note from Fig. ing Fig. . This maequation  for two-45. In monolayer graphene, the temperature dependence of the resistivity near the Dirac point appears to arise predominately from scattering from screened Coulomb impurities, whereas at higher densities it shows signatures of acoustic and optical phonons. In bilayer graphene, short-range impurities dominate the temperature-dependence of the resistivity for nearly all densities. The exception to this behaviour arises near the Dirac point, where thermal activation out of charge puddles becomes significant. A key approach here has been the application of differential-conductance measurements to suppress the influence of quantum corrections, which has allowed us to identify the various transport regimes relevant to these materials. This approach is expected to be broadly applicable to other low-dimensional materials, and should find application, for example, in the study of transition-metal dichalcogenides and topological insulators.In conclusion, in this article we have explored the contributions to the resistance of monolayer and bilayer graphene transistors. Our studies have revealed transitions between different regimes of scattering, as a function of density, temperature, and of the material system, the details of which appear consistent with the predictions of theory2 cap layer, and an underlying, heavily-doped, Si layer that served as a back gate47. Individual graphene flakes were then contacted with Cr/Au (3-/50-nm) electrodes, defined by electron-beam lithography and lift-off. Layer identification was achieved through a combination of optical microscopy and Raman imaging (see Section v) across a region that then served as our channel. Two different kinds of measurement were made in this study; linear conductance (or resistance) was determined by a low-frequency (13-Hz) AC technique, using a small AC signal (vd = 100-μV RMS) as the externally-applied voltage. For measurements of differential conductance (gd), a DC voltage (Vd) of varying amplitude was applied on top of this AC component, allowing us to perform differential-conductance spectroscopy as a function of both Vd and gate voltage (Vg). An important point that must be emphasized here is that, in all figures and associated text, indicated values of Vd are those applied directly to the graphene region under study. These values are determined by utilising the small-signal AC measurements as a calibration; with a knowledge of vd, the external-circuit components, and our measurement of the actual AC voltage drop (v) across the graphene channel, we define a ratio (v/vd) that we use to determine the actual DC voltage drop across the channel (Vd = Vdappl × (v/vd)). In this expression, Vdappl is the DC voltage applied to the outer contacts of the device. Finally, to suppress the influence of quantum corrections, the differential conductance or resistance (gd−1) was measured by applying a small, but fixed, value of Vd and subsequently determining the variation of gd(T)|Vd or gd−1(T)|Vd.Graphene devices were fabricated by exfoliating Kish graphite onto a doped Si substrate with a 300-nm SiOEvaluating the Sources of Graphene’s Resistivity Using Differential Conductance


PD-L1 and PD-L2 gene copy number gains using fluorescence in situ hybridization as well as PD-L1 and PD-L2 expression in 654 patients with resected non-small-cell lung cancer. The prevalence of PD-L1 amplification and polysomy was 3.1% and 13.2%, respectively. The PD-L1 gene copy number status was in agreement with both the PD-L2 and Janus kinase 2 gene copy number statuses. PD-L1 and PD-L2 expression was observed in 30.7% and 13.1%, respectively. Both PD-L1 copy number gains and expression were associated with smoking-related tumors. Tumor cells with PD-L1 genomic gains exhibited significantly higher levels of PD-L1 expression than those without, but PD-L2 copy number gains were not related to PD-L2 augmentation. PD-L1 gene amplification and polysomy were independently associated with PD-L1 expression, with high immune infiltrates and EGFR expression in a multivariate logistic regression model. Comparative analysis between primary tumors and synchronous regional lymph node metastases revealed that the PD-L1 gene copy number alterations were highly consistent and reproducible compared with the PD-L1 expression. Both PD-L1 amplification and level of protein expression were predictors of poor survival using Cox univariate analyses. Therefore, we conclude that an increase in PD-L1 gene copy number can be a feasible alternative biomarker for predicting response to anti-PD-1/PD-L1 therapy.New reliable biomarkers are needed to predict the response to immune checkpoint inhibitors against programmed death-1 (PD-1) and its ligand (PD-L1), because PD-L1 expression on tumor cells has limited power for selecting patients who may benefit from such therapy. Here we investigated the significance of  EGFR) mutations and anaplastic lymphoma kinase (ALK) gene rearrangements, are among the greatest innovations in the field of personalized cancer therapy. However, the outcomes of NSCLC patients have not yet been improved satisfactorily. Lung cancer, of which NSCLC comprises almost 85%, is still a leading cause of cancer-related death worldwide [Treatment strategies for non-small-cell lung cancer (NSCLC) have three main components: surgery, radiotherapy, and chemotherapy. Of these, significant advances in chemotherapy have been made over the past decade, including the selection of new agents based on histology. Molecular targeted therapies for specific genomic alterations, such as epidermal growth factor receptor  is known; this suggests that the activation of JAK-STAT signaling may be partially involved in subsequent changes experienced by cells possessing this focal amplification. In contrast, the prevalence and significance of copy number gains of the specific PD-L1 and PD-L2 loci in NSCLC have yet to be clarified.Copy number gains may be responsible for increased expression levels of genes located at the gained locus in the genome. In primary mediastinal large B-cell lymphoma , HodgkinPD-L1 and PD-L2 genes, as identified using fluorescence in situ hybridization (FISH), were correlated with the upregulation of the corresponding proteins and with patients’ survival outcomes using a large cohort comprising 654 resected patients with NSCLC. In addition, we performed comparative analysis of the gene copy number and protein expression of PD-L1 using specimens of metastatic regional lymph nodes and matched primary tumors, which were obtained from identical surgical resection, to assess consistency and reproducibility of PD-L1 gene copy numbers and PD-L1 protein expression.Here we investigated whether copy number gains of the N = 19; large cell neuroendocrine carcinoma, N = 11; pleomorphic carcinoma, N = 7, large cell carcinoma, N = 5; giant cell carcinoma, N = 2; and carcinosarcoma, N = 1) in 45 (6.9%) cases. PD-L1 was overexpressed in tumor cells in 201 (30.7%) cases. FISH analyses for PD-L1 were successful in 636 specimens. The patient characteristics according to PD-L1 expression and PD-L1 gene copy number status are shown in Table EGFR wild type were significantly higher than among those with PD-L1-negative tumors. Regarding the PD-L1 copy number status, the numbers of cases were 20 (3.1%) for amplification, 84 (13.2%) for polysomy, and 532 (83.7%) for disomy. Polysomy was subclassified into high polysomy in 43 (6.8%) patients and low polysomy in 41 (6.4%) patients. Borderline polysomy was observed in 19 (3.0%) specimens. Among the PD-L1-amplified tumors, the mean value of the PD-L1 signals ranged from 4.5 to 11.9 , and the PD-L1/centromere enumeration probe (CEP) 9 ratios ranged from 2.1 to 4.9 . Among tumors with PD-L1 polysomy, the average PD-L1 signal ranged from 3.0 to 8.6 . Similar to cases with PD-L1 expression, PD-L1 amplification and polysomy were associated with characteristics related to smoking. Interestingly, PD-L1 amplification was not observed among tumors with either mutant EGFR expression or ALK expression.A total of 654 surgically treated patients with NSCLC were included. The tumors were histologically classified as adenocarcinoma in 430 (65.7%) cases, as squamous cell carcinoma in 179 (27.4%) cases, and as other histologies . PD-L2 expression was associated with squamous histology, high immune infiltrates, high EGFR expression, and high p-EGFR expression. PD-L2 amplification and polysomy were observed in 11 (1.7%) and 77 (12.1%) patients, respectively. The mean value of the PD-L2 signals among the tumors with PD-L2 amplification ranged from 4.0 to 9.9 , and that of the PD-L2/CEP9 ratios ranged from 2.0 to 3.9 . The average PD-L2 signal among tumors with PD-L2 polysomy ranged from 3.0 to 7.8 . FISH signals sufficient for the interpretation of both the PD-L1 and the PD-L2 probe sets were observed in 628 tumors. The distribution of copy number alterations of the PD-L1 and PD-L2 genes among those tumors is shown in PD-L1 and PD-L2, which were subclassified into five groups, was 0.91 , 0.87–0.94), indicating excellent concordance. Thus, the clinicopathological characteristics of patients with PD-L2 copy number gains were almost the same as those with PD-L1 copy number increase  specimens, 77 (12.1%) showed polysomy ; low polysomy, N = 43 (6.8%)), and 543 (85.4%) showed disomy ). The mean value of the JAK2 signals among the tumors with amplification ranged from 4.6 to 12.5 , and the JAK2/CEP9 ratios ranged from 2.0 to 4.4 . The average JAK2 signal among tumors with polysomy ranged from 3.0 to 7.5 . FISH signals of both the PD-L1 and the JAK2 probe sets were successfully interpreted in 625 specimens, and their distribution status is shown in JAK2 copy number activated the JAK2/STAT3 pathway by evaluating the phosphorylation status of STAT3 as a surrogate for signaling activity in 634 specimens. However, no association was found among the copy number status of JAK2 and the positivity of phospho-STAT3 (p-STAT3) expression .Of 636 specimens for which PD-L1 are shown in Figure PD-L1 disomy had no or low levels of PD-L1 expression, with a median H-score value of 0 (interquartile range (IQR), 0–2). The expression levels of PD-L1 among tumors with PD-L1 borderline polysomy ), low polysomy ), high polysomy ), and amplification ) were significantly greater than those with PD-L1 disomy. In a multivariate logistic regression analysis (Table PD-L1 amplification (odds ratio (OR), 14.20; 95% CI, 3.90–52.00) and PD-L1 polysomy  were independently associated with PD-L1 expression. High immune infiltrates  and EGFR expression  were also independently associated with PD-L1 expression, and ALK expression tended to be associated with PD-L1 expression . Representative cases with no PD-L1 expression and disomy of both PD-L1 and PD-L2, PD-L1 overexpression and polysomy of both PD-L1 and PD-L2, and PD-L1 overexpression and amplification of both PD-L1 and PD-L2 are presented in PD-L2 copy number gains  paired specimens  for PD-L1-polysomy tumors, and 11.8 years  for PD-L1-disomy tumors. The influence of PD-L1 copy number increase on overall survival was prominent when restricted to PD-L1 expression-positive patients .The estimated median survival time (MST) in the entire cohort was 11.5 years ), with a median follow-up duration of 3.1 years. A significant difference in overall survival was observed between PD-L1-positive and -negative patients , in patients with advanced NSCLC. Significantly, only a subset of patients responded to anti-PD-1/PD-L1 therapy. Biomarkers to predict treatment benefits have been vigorously explored. The nonsynonymous mutation burden, the molecular smoking signature, and the mismatch-repair deficiency of tumors, all of which would result in neoantigen generation, were correlated with the clinical benefits of pembrolizumab [PD-L1 copy number gains were independent and strong predictors of PD-L1 expression. Moreover, PD-L1 copy number status had a higher consistency between regional lymph node metastases and primary tumors, indicating that PD-L1 copy number was sustained less dynamically in cancer cells than PD-L1 expression. In addition, a recent study suggests that PD-L1 amplification might also increase the level of PD-L1 expression in the adaptive state stimulated by immune cells [PD-L1 in NSCLC.New immunotherapies targeting the PD-1 and PD-L1/L2 pathways to reactivate the suppressed tumor immune system have shown promising results in various cancers. In NSCLC, nivolumab, one of the anti-PD-1 antibodies, has been demonstrated to have an encouraging antitumor effect . Among polizumab , 28. Furolizumab , pembrololizumab , and ateolizumab . Howeverolizumab . In triaolizumab , tumor Polizumab  and tempne cells . These rPD-L1 copy number increase were more common among smoking-related NSCLCs. Another study identifies PD-L1 expression more commonly in squamous cell carcinoma than in adenocarcinoma [KRAS-mutant NSCLC. By contrast, Azuma et al. [PD-L1 amplification lost their significant effect on postoperative survival in the multivariate analysis. The prognostic significance of PD-L1 has been variously reported; it may be associated with better [PD-L1 copy number status are needed to clarify the definitive prognostic significance of PD-L1.Our findings showed that both PD-L1 expression and arcinoma . These rarcinoma . Calles arcinoma  report ta et al.  show thah better , 34, 35 h better , 36. In PD-L2 gene. This could possibly be explained by the low affinity of the antibody used for IHC, resulting in lower PD-L2 positivity than PD-L1 and through gene silencing by epigenetic modifications such as DNA methylation and histone deacetylation. As a result, despite the status of copy numbers of PD-L1 and PD-L2 being closely matched, the relationship between PD-L1 and PD-L2 expression was discordant in more than half of the PD-L2-positive specimens. The JAK2 copy number was also co-altered with the PD-L1 copy number. The copy number-dependent JAK-STAT activity is known to induce further PD-L1 expression in lymphoma [PD-L1-amplified lung cancer cell line, HCC4006, is shown to be reduced by JAK2-specific and STAT3-specific inhibitors [JAK2 were not positively associated with expression of p-STAT3 in tumor cells. Further studies are needed in vivo to clarify whether JAK2 is activated in a copy number-dependent manner in NSCLC.In contrast to PD-L1 that is constitutively expressed by antigen-presenting cells, nonhematopoietic cells, and other many organs, PD-L2 expression which is less prevalent than PD-L1 in tumors is relatively restricted to macrophages, dendritic cells, and fibroblasts . Comparelymphoma . In addihibitors . In the EGFR mutations and ALK rearrangements were analyzed in a standard manner, such as PCR-based assays for EGFR and FISH or RT-PCR assays for ALK. We evaluated both gene alterations using IHC methods, which are inferior to standard methods. The sensitivity of IHC is known to be low, especially for the detection of non-15-base pair deletions (E746–A750) in exon 19 for EGFR [EGFR mutation status had been molecularly determined by standard methods were examined, clinicopathological features based on PD-L1 expression and PD-L1 copy number were similar to results from the entire cohort  and 227 patients who underwent surgery between January 2006 and April 2014 at Seirei Mikatahara General Hospital (Japan). All of the patients were Japanese and provided written informed consent for the use of resected specimens for medical research. The study design was approved by the Institutional Review Boards of Hamamatsu University School of Medicine and Seirei Mikatahara General Hospital. The investigation was conducted in accordance with ethical standards and according to the Declaration of Helsinki as well as national and international guidelines. Clinical and pathological data including outcomes were retrospectively obtained from a review of the patients’ medical records. Three board-certified pathologists  classified the lung cancers according to the WHO classification (7th edition). Tissue microarrays (TMAs) in which individual cores had a diameter of 2 or 3 mm were isolated from representative lung cancer tissue, and all the TMA cores were validated to contain a sufficient number of tumor cells by reviewing hematoxylin and eosin (HE)-stained sections, as reported previously , 41. To PD-L1), RP11-635N21 , and RP11-982A21 , were used as locus-specific FISH probes. The spectrum Green-labeled control probe for the near-centromere locus on chromosome 9 (RP11-113O24) (Advanced GenoTechs Co.) was used to enumerate chromosome 9. For nuclear staining, 4′,6-diamidino-2-phenylindole  was used. The probes used in this study had been validated by hybridization to the chromosomal metaphase spread of normal lymphocytes to verify chromosomal numbers and loci. Interpretation of FISH signals was performed without reference to specimen identification. The cores with signals with insufficient intensity for interpretation were excluded from the analyses. After screening the entire area of individual cores, the probe signals for a monolayer of at least 50 tumor cell nuclei were counted at ×100 magnification in at least three representative images per case. The mean values of the target signals  and the CEP9 signals, as well as the mean target BAC signal/CEP9 signal ratios, were calculated. Copy number amplification was defined according to the average target BAC signal/CEP9 signal ratio of ≥ 2.0 , mutant EGFR with the exon 19 deletion or the L858R point mutation, and ALK using IHC, all of which have been previously reported to be associated with PD-L1 overexpression in NSCLC tumor cells , 44, 45.EGFR mutation analyses were performed as part of clinical practice in commercial clinical laboratories (SRL in Tokyo (cycleave method), LSI Medience in Tokyo (peptide nucleic acid-locked nucleic acid PCR clamp method or the cobas EGFR assay), or BML, Inc. in Tokyo (PCR invader method)) for only 267 patients. Therefore, we assessed the EGFR mutation status of all the specimens using IHC with two antibodies specific for the E746-A750 deletion in exon 19 and for the exon 21 L858R mutation. In the present study, the sensitivity, specificity, and positive and negative predictive values for mutant EGFR specific IHC, compared with EGFR mutation status (exon 19 deletions or L858R mutation in exon 21), as determined using the above-mentioned PCR-based methods, were 77.3%, 94.3%, 84.1%, and 91.4%, respectively.Of the 654 recruited patients, U test or the Kruskal–Wallis test for continuous variables were used. P values in multiple comparisons were adjusted using the method of Holm. The extent of agreement was assessed using Cohen's weighted kappa statistics or kappa statistics. Chord diagrams were used to visualize similarities and differences of agreement. Logistic regression was performed to analyze the association between PD-L1 expression and the PD-L1 copy number gains with adjustment for confounders. Overall survival was defined as the time between the date of operation and the date of death or last contact. The survival curves were estimated using the Kaplan–Meier method, and the log-rank test was used to analyze differences in survival time. Cox proportional hazard regression analyses were fitted to determine the impact of risk factors on overall survival with adjustment for other potential confounding factors. The proportional hazard assumption was assessed based on Schoenfeld residuals. Statistical tests were two sided, and P values < 0.05 were considered to be statistically significant. All data analyses were performed using R  and additional packages from the comprehensive R archive network (CRAN) library.The Fisher's exact test for categorical variables and the Mann–Whitney