Anti-immune programmed death-ligand 1 (PD-L1) pathway is used by the tumor to overcome immune system and serves as immunotherapy target in various malignancies.To investigate the expression of PD-L1 in vulvar squamous cell carcinoma (vSCC) and to assess it's clinicopathological and prognostic significance.Immunohistochemical PD-L1 expression was evaluated in 84 vSCCs with previously defined status of p16 and DNA-HPV, infiltration of immune cells: CD8+, CD4+, FOXP3+, CD56+, CD68+, and GZB+ cells. PD-L1 positivity was defined as ≥5% of PD-L1-positive cells. Survival analyses included the Kaplan–Meier method, log-rank test and Cox proportional hazards model.PD-L1 expression was detected on cancer and peritumoral immune cells. PD-L1-positivity of cancer nests  was correlated with higher infiltration of CD4+ (p=0.037), CD8+ (p=0.02), FOXP3+ (p=0.007), CD68+ (p=0.021) cells, while PD-L1 positivity of peritumoral immune cells  was correlated with higher infiltration of intraepithelial FOXP3+ cells only (p=0.037).PD-L1-positivity of cancer cells but not immune cells, was more frequently observed in p16-negative tumors (p=0.004). High-risk HPV-status did not correlate with the PD-L1 status of cancer and immune cells (p=1.000) and (p=1.000) respectively). Median follow up was 89.20 months (range 1.7-189.5). PD-L1 positivity of peritumoral immune cells was found to be an independent favorable prognostic factor for OS. Conclusion: This study highlights the importance of comprehensive PD-L1 assessment in both cancer and immune cells. PD-L1 expression on peritumoral immune cells seems to be an additional prognostic factor in vSCC patients and may influence the results by anti-PD-L1 treatment. The immune microenvironment of vulvar squamous cell carcinoma (vSCC) has been studied intensively by our group –3 and thINK4a expression regardless to high-risk HPV-DNA status [INK4a-negative tumors.Recently, we have found that cancer immune surveillance as represented by tumor infiltrating lymphocytes (TILs) and tumor associated macrophages (TAMs) depends on p16A status  as varioINK4a overexpression, contributes to shaping of the tumor microenvironment and p16INK4a-status could stratify patients for separate immunotherapeutic approaches in vSCC.This suggests that not HPV infection itself, but p16Recently, there has been a breakthrough in cancer immunotherapy against various cancer types by employing immune checkpoint blockade, particularly using antibodies directed against programmed death-ligand 1 (PD-L1) pathway members .PD-L1 , which is expressed on many cancer and immune cells, plays an important part in blocking the ‘cancer immunity cycle’ .INK4a and high risk (hr) DNA-HPV status in the primary tumor.This study aimed to evaluate PD-L1 expression in vSCC tumors and to look for the correlation of this biomarker with clinical and pathological features of vSCC patients including TILs, TAMs as well as p16PD-L1 expression was detected on cancer cells (CC-stars) and tumor-infiltrating immune cells (IC-arrows)  PD-L1 positive CC were found while PD-L1 positivity in peritumoral immune cells was disclosed in 51 of 84 (60.7%) cases.Lack of PD-L1 expression (CC-PD-L1 and IC-PD-L1 negative tumors) was observed in 26 of 84 (31%) vSCC. Dual expression of PD-L1 (CC-PD-L1 and IC-PD-L1 positive tumors) was notified in 20 of 84 (23.8%) cases. PD-L1 was exclusively expressed on cancer (CC-PD-L1 positive and IC-PD-L1 negative) and immune cells (CC-PD-L1 negative and IC-PD-L1 positive) in 7/84 (8.3%) and 31/84 (36.9%) case respectively. Detected variants of PD-L1 expression are depicted on Figure CC-PD-L1-positivity was correlated with higher intraepithelial infiltration of CD4+ (p=0.037), CD8+ (p=0.02), FOXP3+ (p=0.007), CD68+ (p=0.021) immune cells  (Table CC-PD-L1-positivity was frequently observed in p16-negative vSCCs (p=0.004).CC-PD-L1-positivity was similar in (hr)HPV-DNA positive and negative vSCCs (p=1.000).Lack of difference in IC-PD-L1–positivity was observed in cases with mutually excluding p16 and (hr)HPV-DNA status .CC-PD-L1 and IC-PD-L1-positivity did not depend on pT (Fisher's exact test) (p=0.748 and p=0.158), presence of lymph node metastases (p=0.640 and p=0.656), tumor differentiation grade G1/G2,3 (p=1 and p=0.815), (Mann-Whitney U test) age (p=0.523 and p=0.072) and depth of invasion (p=0.361 and 0.502) respectively (Table CC-PD-L1-positivity did not influence prognosis of vSCC patients (p=0.164) Figure .Patients with primary tumors positive for IC-PD-L1 expression had improved OS compared to IC-PD-L1 negative ones (p= 0.017) Figure .In the next step, the cohort of 84 vSCC patients was divided into four groups based on variants of CC-PD-L1 and IC-PD-L1-positivity: CC-PD-L1(+)/IC-PD-L1(-) n=7; CC-PD-L1(+)/IC-PD-L1 (+) n= 20; CC-PD-L1(-)/IC-PD-L1(+) n=31 and CC-PD-L1(-)/IC-PD-L1(-) n=26.Survival analyses comparing all variants of PD-L1 expression indicated the group of patients with CC-PD-L1(-)/IC-PD-L1(+) tumors presented trend towards significantly better prognosis (p=0.061) Figure . When thINK4a status.Additionally, impact of PD-L1-positivity on survival was assessed in groups of patients with different p16INK4a-negative and p16INK4a-positive (p=0.3763 and p=0.1639 respectively).CC-PD-L1-positivity did not influence the overall survival in cases having tumors p16INK4a-positive tumors (p=0.0271) while it was not prognostic for patients with p16INK4a- negative tumors (p=0.0821) . Compared with the whole tumor sections, tumor microarrays (TMAs) may be less representative, especially while assessing the biomarkers expressed in the tumor infiltrating immune cells .PD-L1 positivity was defined as ≥5% of cells regardless the intensity of membranous staining. This is consistent with several publications analyzing this biomarker within other malignancies –12. An aPD-L1-positivity of immune cells was more frequently observed within vSCC than PD-L1-positivity of cancer cells (60.7% vs. 32.1%). We found only two publications dealing with PD-L1 in vulvar cancer , 14. NonINK4a-overexpression modulates immune cells infiltration regardless to (hr)HPV-DNA status [INK4a-negative tumors although more infiltrated with TILs  had worse outcome than cases with p16INK4a-positive vSCCs [INK4a-negative vSCCs suggesting that anti-immune programmed death-ligand 1 (PD-L1) pathway is at least partially responsible for worse outcome in these patients. However, CC-PD-L1 did not reveal prognostic significance either in entire cohort nor in patients with p16INK4a-negative tumors.Recently we have reported that p16A status . Patientve vSCCs , 15. HerInk4a-negative tumors which were found to be more infiltrated by TILs and TAMs [The mechanism by which CC-PD-L1 surface expression is induced is quite ambiguous and includes either immune cell and cancer cells. Besides adaptive PD-L1 up-regulation in an inflammatory cytokine milieu caused by TAMs and/or Interferon-γ –18, cancand TAMs . Lack ofInterestingly, PD-L1-positivity of immune cells was found to be independent prognostic factor for overall survival of vSCC patients. Similar findings were published for head and neck cancer patients suggesting that comprehensive assessment of PD-L1 expression could bring an information for more precise usage check point inhibitors . Recent Several previous reports have shown that PD-L1 expression is a negative prognostic factor in several cancer types, including renal, colorectal, and lung cancers –26, but INK4a-status of the primary tumor, this protective effect was observed only for p16INK4a-positive cases. (hr)HPV-DNA status did not influenced PD-L1-positivity either on cancer and immune cells which supports our previous results suggesting lack of clinicopathological significance of HPV-DNA positivity within vSCC [Notably, we have found that IC-PD-L1-positivity improves survival in entire cohort, but when the patients were subdivided into two groups based on p16hin vSCC , 15.When we separately analyzed PD-L1-positivity on CC and IC, which was categorized into four variants of vSCC cases, we found that patients with CC-PD-L1(-)/IC-PD-L1(+) tumors had significantly best outcome.Few hypotheses could explain this paradoxical favorable prognosis of PD-L1 expression on ICs. IC-PD-L1 expression could be driven by adaptive mechanisms such as exogenous inflammation mediated immune attack and then reflected pre-existing immunity , 30. IC-Here, IC-PD-L1-positivity was correlated with unfavorable intraepithelial FOXP3+ lymphocytes while CC-PD-L1-positivity was correlated with favorable CD4+, CD8+ as well as unfavorable FOXP3+and CD68+ immune cells. Thus, indication of more favorable pre-existing immunity remains unclear.INK4a and (hr)HPV-DNA status. Such correlations have never been described before.However, with the exception of PD-L1 expression all other data were retrieved from our previous studies, originality of current report is supported by completely new study project and design. Immunohistochemical PD-L1 expression has never been analyzed before within vulvar cancer tissue. By analyzing all available data for this cohort, we were able to present in single publication, not only patterns of expression and prognostic significance of this biomarker but also associations with TILs, TAMs, p16This study provides a new insight into immune surveillance on vSCC as for the first time demonstrates that although PD-L1 could mediate the occurrence of cancer immune escape, it also indicates an effective immune response.The weaknesses of the current study are the retrospective design and the small size of cohort involved. Its strength lies in the consistency of the treatment of patients under uniform standards and their long observation, revealing recurrences and hence enabling the assessment of the prognostic significance of all the analyzed biomarkers.This retrospective study was approved by the Polish Ministry of Science and Higher Education review board, who determined that further informed consent was not required as informed consent for tissue sampling was obtained from all patients prior to surgical treatment and written consent was given by the patients for their information to be stored in the hospital database and used for research.INK4a and (hr)HPV status[Our cohort of 85 patients previously studied with known p16PV status, as wellINK4a and (hr)HPV status were retrieved from our recent study [We analyzed all 84 primary tumors for PD-L1 expression. Data on TILs were retrieved from our previous studies , 5. Datant study .Mouse anti-human monoclonal antibody against PD-L1  was obtained from Dako Inc. Mouse anti-human monoclonal antibodies against CD4 (NCL-L-368), CD8 (NCL-L-295), CD56 (NCL-CD56-1B), were obtained from Novocastra, Inc. Mouse anti-human monoclonal antibody against FOXP3 (cat. No ab20034) and CD68 (cat. No ab955) were obtained from Abcam, Inc. Mouse anti-human polyclonal antibody against Granzyme B (cat. No 760-4283) was obtained from Ventana Medical Systems, Inc. Mouse anti-human monoclonal antibody against p16 (cat. No sc-56330) was obtained from Santa Cruz Biotechnology Inc., USA [Immunohistochemical staining for PD-L1 was performed as follows. Four-micron-thick serial sections were cut, placed onto slides, and deparaffinized. For epitope retrieval, slides were immersed in Target Retrieval Solution  and heated in a pressure cooker. Then, they were incubated for 90 min with primary antibody . The reaction was visualized using the EnVision FLEX (DAKO). Appropriate positive (tonsil) and negative  controls were included in each staining. Immunohistochemistry results were evaluated by two independent pathologists blind to the clinical data.PD-L1 cell expression was referred as positive if membranous staining was identified and showed a continuous honeycomb pattern. Cytoplasmic staining of PD-L1 was disregarded. Expression of PD-L1was categorized into two groups according to the percentage of PD-L1-positive cells. PD-L1 positivity was defined as ≥5% of cells regardless to intensity of membranous staining –12.INK4a status was previously described for this cohort by our group [Immunohistochemical staining for other antibodies: p16, CD8, CD4, FOXP3, CD68, CD56, GZB with evaluation and classification of TILs and p16Tissue dissection and DNA preparation as well as mucosal HPV DNA amplification and genotyping were detailed for this cohort in our other recent study .The statistical analysis was performed using the Fisher's exact probability test. The difference between numerical variables was verified by Mann-Whitney U test.p values <0.05 were regarded as significant in all analyses.Overall survival curves were estimated by the Kaplan–Meier method and compared by the two-sided Fox test. Univariate and multivariate analysis was performed using Cox regression model. All analyses were performed using the statistical softwareStatistical 13 (Stat Soft Inc.).PD-L1 expression on IC, not on CC, is independent predictor of favorable OS in surgically treated vSCC patients. Assessment of the expression of immune-related molecules in whole tissue section slides could produce evidence relevant to the appropriateness of treatment via immune checkpoint blockade.Moreover, our findings highlight the importance of comprehensive assessment of both TC and IC in vSCC and suggest that usage of antibodies directed against PD-L1-pathway seems to be favorable for subjects having tumors negative for PD-L1 peritumoral immune cells. Thus, incorporating peritumoral immune cells into the classification of PD-L1 expression seems to be necessary to select the beneficial vSCC patients for anti-PD-L1 treatment.


Linear Dirac dispersion relation is nearly preserved and the calculated small effective mass in the order of 0.05mo suggests high carrier mobility. Density of states and space charge distribution of the considered heterobilayer structure near the conduction and the valence bands show unsaturated π orbitals of stanene. This indicates that electronic carriers are expected to transport only through the stanene layer, thereby leaving the h-BN layer to be a good choice as a substrate for the heterostructure. We have also explored the modulation of the obtained band gap by changing the interlayer spacing between h-BN and Sn layer and by applying tensile biaxial strain to the heterostructure. A small increase in the band gap is observed with the increasing percentage of strain. Our results suggest that, Sn/h-BN heterostructure can be a potential candidate for Sn-based nanoelectronics and spintronic applications.The structural and electronic properties of stanene/hexagonal boron nitride (Sn/h-BN) heterobilayer with different stacking patterns are studied using first principle calculations within the framework of density functional theory. The electronic band structure of different stacking patterns shows a direct band gap of ~30 meV at Dirac point and at the Fermi energy level with a Fermi velocity of ~0.53 × 10 In fact, stanene, a buckled honeycomb structure of 2D hexagonal tin film, has drawn keen interest due to its intriguing prospect as quantum Hall insulator2, topological insulator3 and topological superconductor4. In addition, stable low buckled form of stanene7, synthesized on the Bi2Te3 (111) substrate8 is theoretically investigated to support a large-gap 2D quantum spin Hall (QSH) state at room temperature and enables superior electric conduction with zero dissipation10. However, stanene has a zero band gap without spin orbiting coupling (SOC) while ~0.1 eV SOC induced band gap has been reported in the literature11. This zero band gap feature limits the use of stanene in the high performance semiconductor based nanoelectronic devices such as field effect transistor12. In this context, Garg et al.13 has recently reported the opening of a band gap in stanene by the patterned boron-nitride (BN) doping in stanene using density functional theory (DFT). In addition, Chen et al.14 has studied graphene-stanene heterobilayer models with several stacking methods and reported a band gap of ~77 meV for the most stable configuration. Furthermore, graphene/hexagonal boron nitride (graphene/h-BN) bilayer has been reported to show a small band gap at Dirac point in contrast to the zero band gap of graphene monolayer as well as higher carrier mobility than the graphene bilayers17. In fact, chemical stability and significant band gap of the monolayer h-BN18 make it promising for heterostructure based nano-devices.Of late, group-IV two dimensional (2D) materials such as silicene, germanene and stanene have received special attention attributed to their similar electronic properties with the graphene and intriguing prospects in the nanoelectronic device applications5, we report the structural and electronic properties of stanene/hexagonal boron nitride (Sn/h-BN) heterobilayer using first-principles density functional theory (DFT) calculations. A band gap opening along with the appearance of nearly linear relation near the Dirac point is observed in the proposed heterobilayer structure. Three representative models of the hybrid structure based on different stacking are investigated in terms of their stability, electronic property such as band structure, density of states, real space charge density and effective mass as well as the modulation of band gap under tensile strain and varying the interlayer distance. Our proposed structures with a finite band gap and high carrier mobility would provide further insight and encouragement on the modeling of stanene-based nanoelectronic and spintronic devices.Hence, following the deep scientific interest in modulating the band gap and other electronic properties while preserving the Dirac cone of stanene19. The electron-ion interactions are accounted using norm conserving Troullier Martin pseudo-potentials20. To describe the electron exchange correlation energy, the generalized gradient approximation (GGA)21 with Perdew-Burke-Ernzerhof (PBE) exchange correlation functional is implemented. In order to consider the Van der Waals (vdW) inter-molecular attractive forces, we have used DFT-D222, an empirical dispersion correction proposed by Grimme23 throughout the simulations. For the structural optimization, the plane wave basis cut off is set at 550 eV (40 Ry) with a convergence threshold on force of 10−4 Ry/a.u. The first Brillouine zone of the unit cell is sampled with a 12 × 12 × 1 Monkhorst-Pack grid for the geometry optimization and a 15 × 15 × 1 grid for the subsequent calculations. The interaction between the two adjacent bilayers is eliminated by introducing a sufficient vacuum of 20 Å along the direction perpendicular to the surface.The electronic properties of the proposed heterobilayer structure are investigated using the density functional theory (DFT) with a plane-wave basis set using the Ab initio code PWSCF package of Quantum Espressom* is the particle effective mass, E(k) is the dispersion relation, k is the wave vector and ћ is the reduced Planck constant.The effective masses of electrons 24 and simulation27 studies. This is also in close agreement with the reported theoretical studies by Liu et al.11, Nissimagoudar et al.28 as well as by Peng et al.29. Again, in our study after the geometry optimization the lattice constant of h-BN is 2.504 Å, which is also in close agreement with the previously reported studies33 in the literature. The optimized lattice constant of stanene is ~47% greater than that of h-BN and therefore we have considered stanene/h-BN heterostructures composed of 4 × 4 lateral periodicity of h-BN and 2 × 2 lateral periodicity of stanene, as shown in Fig. In our study, the geometry optimized lattice constant, Sn-Sn bond length and buckling height of the stanene are 4.687 Å, 2.834 Å and 0.82 Å respectively, which is in good agreement with the reported experimental2 heterobilayer structures35. A 4 × 4 supercell of MoS2 with a 5 × 5 supercell of graphene was considered due to the 23% difference in the lattice constants between graphene (2.42 Å) and MoS2 (3.12 Å) and the graphene layer was further stretched by ~3% to meet the commensurability criterion. Also, Chen et al.14 investigated the electronic and optical property of graphene/stanene heterobilayers where the lattice constants of graphene and stanene differ by about 46%. Hence, they14 proposed a supercell composed of 4 × 4 graphene and 2 × 2 stanene and further stretched the stanene layer by 4.7% to form commensurable structures.The basic unit cell consists of two Sn atoms, four B atoms and four N atoms. Our Sn/h-BN heterobilayer configuration can be compared to the reported literature on the graphene/MoS36, graphene on copper substrate36, germanene/BeO37 and graphene/stanene14.Three stacking patterns of stanene-hexagonal boron nitride (Sn/h-BN) have been considered in this study as shown in Fig. et al.32 for graphene/h-BN heterobilayer. Our obtained equilibrium distances are also comparable to the reported38 interlayer distances (3.57 Å to 3.75 Å) for bilayer h-BN using GGA in the scheme of PBE. It is also comparable to the experimentally obtained interlayer distance of the Sn bilayers (3.5 Å ± 0.2 Å) during the epitaxial growth of stanene8 as well as with the calculated interlayer distance of the Sn bilayers (3.6 Å) by Evazzade et al.39 using GGA and PBE exchange correlation in the Quantum Espresso package where DFT-D modeling has been employed. On the other hand, vdW-DF2 has been reported to yield an equilibrium interlayer distance higher than the experimentally determined interlayer distance for some heterobilayers (3.77 Å for graphene-SiC32). In fact, the equilibrium interlayer distance is found to be sensitive with the choice of the exchange-correlation functionals40.Figure 15.Moreover, as suggested by the binding energies of the three configurations shown in Fig. 41 than the binding energies of the weak vdW interactions42. This indicates that the stanene monolayer is bound to the h-BN substrate via interactions somewhat stronger than the weak vdW interactions43. The heterobilayers such as graphene/h-BN36, stanene/MoS244 and graphene/SiO242 are reported to have weak vdW interactions between the heterobilayers with a binding energy of less than 100 meV per unit cell. On the other hand, in the graphene/HfO2 heterobilayer structures, the graphene is strongly bound to the HfO2 with a binding energy of 110 meV43. The similar phenomenon has been reported in the study of the graphene/stanene heterobilayers14 with a binding energy of more than 180 meV per unit cell at optimum spacing. As an aside, the optimized interlayer distances for the three stacking configurations of the Sn/h-BN heterobilayers considered in this study are 3.7 Å to 3.78 Å, which are larger than the typical bond length of the Sn-B bonds (2.24 Å)13 and the bond length of the Sn-N bonds (2.14 Å)13. This indicates that, the Sn-B and the Sn-N covalent bonds are absent in the Sn/h-BN heterostructures and further conforms to the similar phenomena reported regarding the graphene/stanene14 and the graphene/HfO243 heterobilayers.Furthermore, the calculated binding energies in our study for the Sn/h-BN heterobilayers per unit cell at equilibrium are more than 250 meV which is higheret al.45 (70 meV direct band gap in the presence of SOC) as well as by Xiong et al.46 (73 meV direct band gap with SOC), thereby confirming the validity of our simulations. Furthermore, in the presence of SOC a Fermi-level band crossing phenomenon of bands is observed at gamma (Γ) point. For the band structure of stanene as well as stanene nanoribbons in the presence of SOC, at gamma point similar phenomenon of either bands touching or crossing the Fermi level has been reported by Broek et al.5, Modarresi et al.45, Lu et al.47, as well as by Nagarajan et al.48. Despite this phenomenon, in view of the opening of the band gap at K point Broek et al.5 concluded that, pristine stanene offers perspectives of an all-Sn field effect transistor (FET). However, as discussed by Xiong et al.46 and Nagarajan et al.48, the effect of SOC on the band structure of stanene and the Fermi level touching or crossing phenomenon indicates its possibility of applications in spinelectronics and quantum spin Hall fields. Hence, the overall effect of SOC in the band structure of stanene and stanene based nanostructures requires consideration in terms of their promising application in nanoelectronics, spinelectronics, quantum spin Hall insulators as well as field effect transistor.For further benchmarking, we have computed the band structure of the 2D monolayer stanene without SOC as well as in the presence of SOC as shown in Fig. Figure 49. However, at gamma point (Γ) a Fermi-level band crossing phenomenon of bands can be observed which is similar to pristine stanene with the effect of SOC indicating the possibility of applying Sn/h-BN heterobilayers in the application of spinelectronics and quantum spin Hall insulators48.With the inclusion of SOC, the observed band gap for the structure I, II and III are 74 meV, 109 meV and 90 meV, respectively as shown in Fig. mo respectively, which are small enough to provide high carrier mobility. The computed electron effective masses for the other two configurations are 0.054  × 106 ms−1, 0.526 × 106 ms−1 and 0.538 × 106 ms−1 for structure I, II and III, respectively which are in the similar order of magnitude to that of stanene5 (0.97 × 106 ms−1) and graphene50 (106 ms−1).The linear Dirac disperison near K point suggests a high charge carrier mobility. To further analyze this fact, we have calculated the effective mass of electrons et al.51 for the stanene/MoS2 heterostructure where the strong interaction breaks the sublattice symmetry of stanene and opens a band gap. Similarly, the strong interaction with the BeO monolayer substrate introduces a finite band gap in the germanene/BeO heterostructure by breaking the symmetry of the germanene37. Also, due to the symmetry breaking of the two carbon atoms in the hexagonal graphene induced by the interaction with the h-BN monolayer, a finite band gap of ~50 meV opens as reported in the literature36.Due to the interaction between the stanene and the h-BN layer, the sublattice symmetry of the zero band gap hexagonal stanene structure is broken, therefore opening a direct band gap. Similar phenomenon has been reported by Xiong 5. Furthermore, as shown in Fig. 52, h-BN orbitals do not hybridize with stanene orbitals near the Fermi level ensuring non-intensive interactions of the two layers. This is further supported by the real space charge density distribution of the conduction and valence bands for structure III as shown in Fig. et al.18, the band structure for the graphene/BN bilayer is dominated by the bands associated with the carbon atoms near the Fermi level, with a band gap of ~100 meV at the Dirac point. Similarly, silicene is reported53 to dominate the electronic transport in the silicene/h-BN heterobilayers and the Dirac cone of the silicene layer is preserved after contacting the BN layer, which is in accordance with our study for the stanene/h-BN heterobilayers. In fact, the heterostructures consisting of single layer silicene and graphene sandwiched between h-BN bilayers also show a band gap opening and high carrier mobility while the silicene and graphene layer remain nearly unaffected by h-BN55. In addition, the periodic heterostructures with a graphene sheet sandwiched between four h-BN layers also show similar phenomenon56.Next, to analyze the electronic properties and gain deeper insight on the interlayer interactions, we have plotted the total as well as the projected density of states (PDOS) for structure III of the stanene/h-BN heterobilayer as shown in Fig. In this section, we focus our study to tune up the band gap of the Sn/h-BN heterostructure for its possible use in the high performance nanoelectronic and spintronic devices.58 therefore reducing the band gap. This is in line with the study of graphene/h-BN heterobilayers15 of various stacking patterns where the band gaps increase with the decrease in interlayer spacing.Firstly, we have calculated the energy band gaps with the variation of the interlayer spacing of the stanene and h-BN layers for all three considered configurations as presented in Fig. 5. On the other hand, the reverse phenomenon i.e. the upward shift of Fermi level is expected for the compressive strain.Figure Finally, we have investigated one non-high symmetry configuration for the Sn/h-BN heterostructure as shown in Fig. et al.36.The electronic band structure for the non-high symmetry configuration is shown in Fig. In summary, we have investigated the structural and electronic properties of the Sn/h-BN heterobilayers from DFT studies considering van der Waals interaction between two layers. We have considered three different stacking patterns and all of these are found to be electrically stable in terms of the high binding energy. The band structures of the Sn/h-BN heterobilayer shows a direct band gap of about 30 meV at the Fermi energy level while linear Dirac dispersion relation is closely maintained. The real space charge distribution and density of states of the heterostructure suggest the localization of stanene π orbital in the conduction as well as in the valence bands, further confirming the superior role of stanene in shaping the electronic properties of the stanene/h-BN heterostructure. Moreover, our calculated small effective mass and the emergence of Dirac cone suggest high charge carrier mobility for the Sn/h-BN heterostructure. Furthermore, our analysis concludes that, the band gap can be effectively tuned by varying the interlayer spacing between the Sn and h-BN monolayer while preserving the stability. A small increase in the band gap is observed with the increasing percentage of tensile biaxial strain. At a higher percentage of strain, the Fermi level shifts downward and self hole doping characteristics with hole carriers appear. Such unique and tunable electronic properties of the Sn/h-BN heterobilayer would further encourage the Sn-based nanoelectronic and spintronic device applications.


Graphene is an excellent modifier for the surface modification of electrochemical electrodes due to its exceptional physical properties and, for the development of graphene-based chemical and biosensors, is usually coated on glassy carbon electrodes (GCEs) via drop casting. However, the ease of aggregation and high defect content of reduced graphene oxides degrade the electrical properties. Here, we fabricated low-defect graphene electrodes by catalytically thermal treatment of HPHT diamond substrate, followed by the electrodeposition of Au nanoparticles (AuNPs) with an average size of ≈60 nm on the electrode surface using cyclic voltammetry. The Au nanoparticle-decorated graphene electrodes show a wide linear response range to vanillin from 0.2 to 40 µM with a low limit of detection of 10 nM. This work demonstrates the potential applications of graphene-based hybrid electrodes for highly sensitive chemical detection. Vanillin  is the flavor component of vanilla bean, which is widely used in foods, beverages, and medicines ,2, becau2-bonded carbon atoms. It has extensive influence in the field of electrochemistry due to its exceptional electrical properties, like high carrier mobility ) and high specific surface area (2600 m2/g) [Graphene is a two-dimensional material composed of hexagonally constructed sp00 m2/g) ,17,18,1900 m2/g) ,21,22,2300 m2/g) . Moreove00 m2/g) .Compared to rGO, chemical-vapour-deposited (CVD) graphene exhibits lower electrical resistance and fewer structural defects. However, the lack of functional groups on the surface of CVD graphene limits its electrochemical activity. Therefore, it is efficient to improve the device sensitivity based on CVD graphene via further surface modification, such as molecular doping, the grafting of functional groups, and the decoration of noble metal nanoparticles ,27,28. R3-to-sp2 conversion process. Then, AuNPs were electrodeposited on the graphene surface using a simple cyclic voltammetry (CV) method. The fabricated electrode exhibits a linear electrochemical detection range of 0.2–4 µM with a low detection limit of 10 nM, demonstrating potential applications of graphene-based hybrid electrodes for highly sensitive chemical detection.In this work, we fabricated graphene-diamond hybrid electrodes by a catalytic annealing process using a thin Ni layer as catalyst. High-quality graphene films were formed directly on the diamond surface through an in-situ sp2O2, H2SO4, CuSO4, KCl, HCl, Na2HPO4, NaH2PO4, and vanillin were purchased from Sinopharm Chemical Reagent Co. Ltd. Shanghai, China.High-pressure and high-temperature (HPHT) diamonds with dimensions of 3.5 × 3.5 × 1 mm were purchased from Shenzhen Tiantian Xiangshang Diamond Co. Ltd., Shenzhen, China. Nickel (99.995%) was brought from New Material Technology Co. Ltd., Beijing, China. H2SO4:H2O2 = 7 mL:3 mL) solution at 50 °C for 4 h to remove surface contaminants, followed by ultrasonic cleaning in deionised water and ethanol for 10 min, respectively. A 20-nm-thick Ni film was deposited on the diamond surface via an e-beam system  at a base pressure and deposition rate of 1.4 × 10−5 Torr and 0.5 Å/s, respectively. The Ni/diamond substrate was then thermally treated at 1020 °C for 15 min in a tube furnace system , with H2 flow of 8 sccm. Finally, the graphene–diamond hybrid electrodes were obtained after Ni removal in an etching solution (10 g CuSO4 and 50 mL HCl in 50 mL deionised water). The graphene–diamond hybrid sample was immobilised on a plastic substrate and connected by silver paints to silver wire. Except for the graphene surface, other exposed areas were protected by silicone resin. Au nanoparticles were then electrodeposited on the graphene surface via CV at a scan rate of 100 mV/s with 0.2 mL of 1% AuCl4 solution diluted in 10 mL PBS (PH = 7.0) solution with deposition times varying from 2 to 16 min.The HPHT diamond substrate used in this study was comprised of (1 0 0) singe-crystal diamond. The diamond substrate was first immersed in a mixture of sulfuric acid and hydrogen peroxide  and X-ray photoelectron spectroscopy . The surface morphology was observed using a field emission scanning electron microscope . The electrochemical experiments were carried out with an Autolab workstation . The mercurous chloride reference and Pt counter electrode were obtained from Aida Hengsheng Co. Ltd., Tianjin, China.3-to-sp2 conversion process, in which the HPHT diamond was used as a carbon source. The sp3-bonded carbon atoms on the diamond surface were dissolved into a thin nickel film (≈20 nm) at high temperature (1020 °C) and then precipitated from the nickel film to form sp2-bonded carbons during a rapid cooling process. The success of sp3-to-sp2 conversion can be demonstrated by XPS analysis, which was used to determine the bonding information of the diamond surface before and after catalytic thermal treatment. As shown in 3 bonds (C–C), while the ratio of sp2–sp3 bonds of the graphene–diamond surface increased to 1:1 after annealing. The quality of the obtained graphene was investigated with the Raman spectrometer, as shown in −1, 1586 cm−1, and 2698 cm−1 can be assigned to the D-band, G-band, and 2D-band of graphene, respectively. The I2D/IG ratio (≈0.62) and full width at half maximum (FWHM) of the 2D-band (≈64.5 cm−1) suggest that the graphene is composed of few layers [D/IG ratio (<0.1) reflects that the graphene converted from diamond has low defect content [−1, which can be assigned to the single-crystal diamond [2D/IG ratio of graphene film mostly varied between 1.0 and 1.5, showing that the diamond surface was covered by few layers of graphene [As schematically illustrated in w layers . In addi content . We noti diamond , confirmgraphene .−2 and ≈8 μm−2 for the samples after electrodeposition for 4 and 16 min, respectively, as exhibited in In order to enhance the sensitivity of graphene hybrid electrodes for detecting vanillin, the as-prepared electrode was further modified with AuNPs via electrodeposition. The mechanism of AuNP-decorated graphene electrodes for vanillin detection was investigated by changing the scan rate of CV measurement. As shown in ed works ,21. Our 3-to-sp2 conversion process, the graphene–diamond hybrid electrodes were fabricated via catalytic thermal treatment. The low-defect graphene electrodes were further modified by electrodeposition of AuNPs using the CV method, and the deposition time was optimised to 4 min for achieving a good trade-off between the size (≈60 nm) and surface density of AuNPs, resulting in the highest electrochemical sensitivity for vanillin detection. As a result, a wide linear dynamic range of the hybrid electrodes can be achieved from 0.2 to 40 µM with a very low limit of detection of 10 nM. The AuNP/graphene sensing platform is highly efficient for vanillin determination in food, cosmetic, and pharmaceutical analysis.Based on an sp


This performance seems not to be connected to the strong orbital hybridization that Ni and graphene interface present, indicating electron transfer might not be playing a main role in the robust response of this nanostructured system. The observed protection from biological environment can be understood in terms of graphene impermeability to transfer Ni2+ ions, which is enhanced for few layers of graphene grown on Ni. We expect our work will provide a new route for application of graphene as a protection coating for metals in biological environments, where current strategies have shown short-term efficiency and have raised health concerns.In this work we present a study on the performance of CVD  graphene coatings grown and transferred on Ni as protection barriers under two scenarios that lead to unwanted metal ion release, microbial corrosion and allergy test conditions. These phenomena have a strong impact in different fields considering nickel  is one of the most widely used metals in industrial and consumer products. Microbial corrosion costs represent fractions of national gross product in different developed countries, whereas Ni allergy is one of the most prevalent allergic conditions in the western world, affecting around 10% of the population. We found that grown graphene coatings act as a protective membrane in biological environments that decreases microbial corrosion of Ni and reduces release of Ni When metals are in contact with biofilms, which naturally form under the exposure of materials to ambient moisture, miscellaneous metabolic activities lead to the production of organic and inorganic acids, volatile compounds and other chemical reactions with the metals. These processes induce a highly accelerated deterioration of the substrate known as microbiologically influenced corrosion or microbial corrosion . This phCurrent strategies to prevent microbial corrosion in metallic structures aim to attack biofilms that cause microbial corrosion. They mainly include physical methods  and chemical methods  ,4. The u2+ ions from Ni metallic objects that come in direct contact with human skin has been related to a phenomenon known as the allergic contact hypersensitivity response to Ni [On the other hand, there is a phenomenon that, similar to microbial corrosion, includes metal ion release when a metallic material is in contact with other living systems, such as the one provided by human skin. Among metallic materials, Ni and its alloys are one of the most widely used in diverse domestic objects that come in contact with human skin. Ni has been chosen for most of these applications due to its mechanical properties, corrosion resistance, durability, appearance and availability. However, remarkable as these properties are, they do not seem to withstand direct interaction with human skin conditions. Release of Nise to Ni ,7. This se to Ni ,12,13,142+ is not only released from coins [2+-induced contact dermatitis [2+ ions have been reported to trigger an innate immune response in resident skin cells [Niom coins  but alsormatitis ,17,18. Ain cells  they arein cells . When niin cells . This acin cells ,22. Reduin cells ,24, whicin cells ,26. Evenin cells .2+, by capturing nickel ions [Different nanoscale approaches have been used to produce certain degrees of control over contact allergy and microbial corrosion issues. For instance, the application of calcium phosphate and calcium carbonate nanoparticles to the skin seems to be effective in preventing the allergy towards Nikel ions , which akel ions ,30. Simikel ions ,32. HoweRecently our group and others have reported Cu microbial corrosion passivation using single-layer CVD graphene (SLG) grown on that metal ,33,34. AMotivated by the need of studies exploring the protection that graphene coatings offer to other relevant metals (other than copper), we present a study on the efficiency of graphene grown and transferred on Ni as an ionic barrier under two biological conditions, microbial corrosion and sweat immersion. Chemical vapor deposition (CVD) graphene, with surface areas in the centimeter square range, has been chosen as coating due to its versatility and lack of intrinsic cytotoxicity . This coIn this work we explored the efficiency of as-grown few layer graphene (FLG) and transferred graphene onto Ni foils. We mainly focus on the way in which these nanostructured coatings modify interaction between: (1) Ni and bacteria  and (2) Ni and sweat .−5 hPa whereas during graphene growth was kept at 1.2 × 10−2 hPa. A slight modification of this methodology is introduced by supplying this mixed flux in five steps of 20 min each. Between each step, the sample was held only under the hydrogen flux for 10 min to ensure a high coverage. The final methane step was followed by rapid cooling under a hydrogen-argon flux of 10 sccm and 20 sccm respectively. Similarly, few-layer graphene grown on nickel was obtained through methodology reported in ref. [Graphene grown on Ni and graphene transferred onto Ni surfaces were included in this study to establish a comparison between performances of these graphene coatings obtained through different methodologies. The performance of graphene transferred on Ni will allow a comparison between the same type of nanometric membrane but with different electronic interaction (hybridization) with metallic substrate; weak coupling for graphene transferred on Ni and strong for grown samples ,36. Grap in ref. , using aSingle-layer graphene was transferred onto Ni substrate using the PMMA-assisted method . For thi2 surface area were used for all measurements.Corresponding Ni control substrates were prepared treating fresh Ni (NiF) foils  under the same temperature and hydrogen pressure conditions used for graphene growth but without the carbon precursor gas. Thermally treated Ni samples (control samples) will be called NiT. Samples of 1 cm−10 torr), samples were annealed at 100 °C for 20 min to remove all gases adsorbed on sample surface. Graphitic quality of as-grown FLG was investigated by MicroRaman measurements  at ambient conditions at low laser power (<1 mW).A combination of scanning electron microscopy (SEM) , scanning tunneling microscopy  and X-ray photoelectron spectrometry  was used to characterize samples morphology and chemical environment. Prior to STM and XPS analysis, where ultra-high vacuum conditions are required  by filtration of the suspension using negative pressure. The filters with a bacteria film were dried at room temperature during 90 min in order to obtain a stable water contact angle measured by sessile drop method using 1 μL of distilled. The contact angle was measured based on image analysis [Surface hydrophobicity of coated and uncoated Ni samples was determined by contact angle measurements. For this characterization a drop of milliQ water (2 μL) was placed on the surface of coated and uncoated samples and images were immediately captured using a high-resolution camera. Bacterial hydrophobicity was measured following standard methods  with somanalysis  using thanalysis .Escherichia coli MG1655 cultures, which have been reported to cause metal microbial corrosion [−1) and yeast extract (5 g·L−1). The bacterial cultures were concentrated by centrifugation , washed three times with Milli-Q water, and finally resuspended up to a turbidity of 3.0 at 600 nm. The turbidity of this stock dispersion is equivalent to a bacterial concentration of ~1 × 109 CFU/mL−1 (Colony forming units per milliliter). This high bacterial concentration was chosen to ensure response of graphene coatings is robust enough, even for large number of bacteria. Milli-Q water was used as dispersant to avoid bacterial duplication and the forthcoming accumulation of mineral crystals that may interfere with the collection of microscopy images or cause unwanted chemical reactions with the samples. Nickel release from metallic samples exposed to bacteria was determined by Inductively Coupled Plasma (ICP) . Control samples (NiT) were exposed to Milli-Q water without bacteria. After 24 h, bacteria were recovered, poured into 2.5 mL of 15 μM EDTA (ethylenediaminetetraacetic acid)  solution (pH 10) and centrifuged at 5000× g during 10 min. The supernatant was recovered, and the total Ni concentration was also quantified by ICP.To explore response of graphene coatings (FLG and 4MLG) to biological environments and their ability to prevent microbial corrosion of Ni, we used orrosion ,52. BactE. coli MG1655 stock dispersion was placed on each sample surface in order to obtain a final bacterial density of 60 μL/cm2. Sample + bacteria systems were incubated at 37 °C during 24 h in a humidity chamber to avoid evaporation. Once this incubation period completed, bacteria were recovered with 3 volumes of Milli-Q water using a standard micropipette. Cell viability at 0 and 24 h was determined using the microdot methodology [v/v) glutaraldehyde and dehydrated by washing with a graded ethanol series (from 10% to 100%), followed by critical-point drying and gold coating.Cell viability (inverse to cell death) was monitored to evaluate the antibacterial activity of coated and uncoated Ni samples. Such antibacterial performance is a direct consequence of Ni ions release and, in our case, will be an indirect indicator of Ni microbial corrosion (in addition to previously mentioned ICP measurements). One volume (100 μL) of hodology . Each exTo study the response of graphene-coated Ni samples to sweat exposure immersion tests were performed for FLG, 4MLG and Ni samples. The artificial sweat solution was prepared similar to EN1811 , using 0Morphological characterization of samples prior to bacterial and sweat contact was obtained by SEM a–d. FresScanning tunneling microscopy in ultrahigh vacuum conditions was used to visualize FLG grown on Ni with nanoscale and atomic resolution e. Charac−1) and 2D (~2688 cm−1) bands, with a small G/2D (~0.23), consistent with literature [−1). Raman spectra displayed typically sharp G (1584 cm−1) and broad 2D (~2701 cm−1) bands for FLG  and FLG coatings, we performed microRaman measurements on these samples. Multiple areas of these samples were analyzed, and representative spectra are shown in terature . Upshift for FLG h, consissharp G 14 cm−1 an3/2 spectrum, which contains information on metallic Ni , NiO (peak in green), and Ni(OH)2 (peak in blue) surface contents that are present when nickel is exposed to ambient conditions [2 signals (in blue) are present for NiF and NiT samples (and to lesser extent for FLG), they are stronger for NiF. This is in agreement with previous reports where an initial Ni oxide growth in metallic Ni saturates at few layers after which oxygen in air leads to formation of Ni(OH)2 on the surface, that continue growing for longer exposure to atmospheric gas pressure [2 at the air-oxide interface and NiO between the metal and hydroxide layer. Nickel metallic signal is stronger for graphene-coated Ni than for bare Ni samples, regardless heat treatment. However, it is clear that NiT samples, due to oxide removal treatment, are similar to graphene-coated Ni in terms of metallic Ni composition, indicating that NiT is in fact the control sample for viability, microbial corrosion and sweat exposure measurements.Surface composition information of Ni samples was obtained by means of XPS analysis a. In parnditions ,60. Stropressure . The resE. coli [Contact angle measurements were performed to characterize surface hydrophobicity of coated and uncoated Ni samples b–d. HydrE. coli , tends tE. coli . In the E. coli . HydrophE. coli . AccordiE. coli after 24 h of incubation on NiT, NiF and graphene-coated Ni samples, together with morphology of bacteria after incubation  exhibit a wide range of significant abnormalities (E. coli on FLG grown on Ni (2)  and the consequent release of Ni ions.SEM images of malities d,e, suchmalities f, in agrwn on Ni b, similan Ni (2) a. This in Ni (2) f. For 4M2 (control) samples indicates that a connection between charge transfer from Ni to bacteria through graphene and their bactericide activity is less likely.Electron transfer from microbial membranes to graphene has been reported to produce a strong antibacterial effect in graphene grown on copper system , where tE. coli cells was chosen as control sample. Solutions with and without bacteria (control) do not contain salts than might affect electrochemical interaction with metal. Dissolved Ni from graphene-coated Ni samples (FLG) in contact with Milli-Q water with bacteria is close to the concentration obtained in control sample. Ni ions release is reduced up to 98.7% when using FLG coating (compare to NiT—control sample of FLG) and up to 46% when using transferred graphene (compare to NiF—control sample of 4MLG). These results demonstrate grown graphitic membranes efficiently protect underlying Ni from microbial corrosion due to bacterial interaction. In particular, Ni dissolution is found to be inversely related to cell viability  while for graphene-coated samples Ni ion release reaches a similar value (~0.6 µg/cm2/week).To study the performance of graphitic coatings under Ni allergy conditions we performed ICP measurements on Ni dissolution for graphene-coated and uncoated Ni samples under sweat immersion conditions  . Nickel ith skin ,22. Our Different coatings have been reported to be used to reduce Ni release from biomaterials in contact with artificial sweat ,70. In v2+ ion release for NiF, when compare with NiT.SEM micrographs of uncoated Ni samples after sweat exposure (insets in 2+ (70 pm) [Our results suggest that the observed effectiveness of graphene as a barrier against interaction between Ni and bacteria or sweat can be understood in terms of graphene permeability. Graphene’s p orbitals form a dense and delocalized cloud that blocks the gap within its aromatic rings , creatin (70 pm) , which a2+ ions release from Cu foils in contact with bacteria [bacteria . The pro2+ ions, which is enhanced for a few layers of graphene.In conclusion, we studied the performance of graphitic coatings as a protection barrier under microbial corrosion and Ni allergy conditions. From the microbial corrosion point of view, cell viability and Ni dissolution results indicate these nanostructured coatings effectively block interaction between bacteria and underlying metal. The absence of bactericide effect for this highly electronically hybridized system suggests that most of the protection from the observed protection from biological environment can be understood in terms of graphene impermeability to transfer NiThis protective effect of impermeable graphene coatings is extended to the case of Ni allergy conditions. Although Ni dissolution does not remain below the value established by international Directive 94/27/EC in graphene-coated Ni samples, its proximity to this value confirms the great potential of graphitic coatings to prevent the significant amounts of nickel ions usually released from metallic devices, which may interact with the human body causing adverse health effects. The most effective way of preventing nickel allergy is the avoidance of exposure. The ideal case would be the complete ban of nickel use, which is irrelevant due the wide distribution of the metal worldwide. In this scenario the observed performance of graphitic coatings to prevent Ni ion release suggests a promising new venue to prevent allergy reactions. Further in vivo experiments are needed to evaluate biocompatibility of graphene-coated materials on humans and animals.


Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs. PD-L1 is upregulated in many cancers due to exogenous cellular stress. Here the authors show that PD-L1 is upregulated in response to DNA double strand breaks via STAT and IRF1 signalling. PD-1 has a role to inhibit T-cell proliferation and interferon-gamma (IFNγ) production in T cells2. Programmed death-ligand 1 (PD-L1) is identified as the ligand of PD-13. The major role of PD-1/PD-L1 interaction is to regulate autoimmune response in the peripheral tissue; PD-1-deficient mice show hyperactivation of the immune system2. Further, mounting evidence showed that PD-1 or PD-L1 deficient mice developed different autoimmune diseases in each genetic background2. Alternatively, the downregulation of PD-1/PD-L1 interaction can be a cause of human autoimmune disease. The association was reported between single-nucleotide polymorphisms (SNPs) on human PD-1 gene or augmented PD-1 expression and human autoimmune diseases4. Thus, the interaction between PD-1 and PD-L1 is critical to control a balanced global immune response in the human body. In addition, PD-1 has roles in cellular response; for example, PD-1 is involved in the immune reaction against HIV infection5.Programmed cell death-1 (PD-1) is an immune receptor, which is expressed on activated CD4+ T cells and CD8+ T cells as well as on B cells in the periphery7. Notably, PD-1 therapy provides significant clinical benefits for patients with an advanced stage cancer11. Despite this therapy having a substantial effect in advanced cancers in many cases, 20–40% of patients still show progressive disease. A recent clinical report described that, in advanced PD-L1-positive non-small-cell lung cancers, anti-PD-1 therapy as a first-line treatment is associated with significantly longer progression-free survival and improved overall survival compared with the standard platinum-based chemotherapy12. In addition, high responders frequently show elevated PD-L1 expression due to gene amplification and upregulation of an ectopic promoter by translocation14. Thus, the evidence suggests that PD-L1 level in tumours is an important factor influencing therapeutic efficacy for responders17, although the correlation is not perfect and the mechanisms underlying this correlation in non-responders have not been precisely revealed18. Recent studies have suggested that cancers with mutations of genes contributing to genomic stability could be targets for anti-PD-1 therapy. Significantly high rates of responses in cancer with microsatellite instability (MSI), which is a hallmark of genome instability, to anti-PD-1 therapy have been reported in colon cancer20. Accumulating studies suggested that MSI-positive tumours presenting neoantigens promote the release of IFNγ from tumour-infiltrating lymphocytes (TILs), and the released IFNγ upregulates PD-L1 expression in immune cells and tumours24. Therefore, PD-1 therapy is considered to be useful for tumours exhibiting high MSI. Consistent with this notion, patients with mismatch-repair (MMR)-deficient cancer showed a higher rate of progression-free survival following anti-PD-1 therapy, demonstrating that MMR status is a potent predictive marker25. High expression of PD-1/PD-L1 in TILs was also observed in DNA polymerase ε-mutated and MSI cancers26. Furthermore, the possible involvement of homologous recombination (HR) repair has been suggested recently; specifically, enrichment of mutations in BRCA2, an HR factor, has been identified in melanomas responsive to anti-PD-1 therapy27. HR deficient tumours were also shown to exhibit greater neoantigen loads, TILs and PD-1/PD-L1 expression in immune cells23. Thus, the evidence suggests that genomic instability causes high levels of mutations and neoantigen loads in tumours, resulting in greater PD-1/PD-L1 expression in cells surrounding tumour microenvironment.Since decades ago, several distinct strategies for cancer immune therapy have been proposed. Among them, anti-PD-1 antibody has been newly developed as a next-generation immunotherapy agent that blocks the immune checkpoint pathway29. Importantly, PD-L1 expression in cancer cells was found to be transiently upregulated following IR, that is, for several days after irradiation, and when the PD-1/PD-L1 interaction was blocked during this upregulation, the anti-PD-1 antibody treatment rescued T-cell activity and delayed tumour growth after irradiation in an immunocompetent mouse model31. Anti-PD-1 therapy was also found to enhance the efficacy in patients treated with carboplatin, suggesting its beneficial effect when used in combination with chemotherapy32. DNA double-strand breaks (DSBs) are the most critical type of DNA damage induced by radio/chemotherapy and activate signal transduction by phosphoinositol-3-kinase-related kinases, ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and DNA-PKcs. Such signalling was previously shown to promote DNA repair and arrest the cell cycle, or to trigger apoptosis. In addition to these functions, previous studies have shown that induction of DNA damage response upregulates the expression of cell-surface molecules, such as NKG2D ligand33. However, so far, the relationship between DSB-dependent damage signal and PD-L1 expression in cancer cells has not been elucidated. In this study, we showed for the first time that DSBs upregulate PD-L1 expression in an ATM/ATR/Chk1-dependent manner. Depletion of BRCA2 enhances PD-L1 upregulation after DSBs. PD-L1 expression in Ku-depleted cells requires Chk1 after Exonuclease1 (EXO1)-dependent resection. Finally, we showed that DSBs activate STAT1/STAT3 phosphorylation and IRF1, and IRF1 is required for DSB-dependent PD-L1 upregulation, i.e. through the canonical pathway. This new understanding of the molecular mechanism underlying PD-L1 expression in cancer cells should aid the development of strategies to predict prognosis and may contribute to improving the efficacy of therapies in DNA-repair-defective cancer cells, particularly when anti-PD-1 therapy and radio/chemotherapy are combined.In cancer treatment, radiotherapy and chemotherapy  are applied to induce lethal DNA damage in cancer cells. Recent studies have shown that ionising radiation (IR) synergistically promotes antitumor immunity when applied in combination with immune checkpoint inhibitorsTo test the responsiveness of PD-L1 expression in cancer cells after DNA damage, we examined PD-L1 protein levels by immunoblot analysis. PD-L1 expression in osteosarcoma (U2OS), lung cancer  and prostate cancer (DU145) cell lines was upregulated after IR, which induces DSBs, in a time- and dose-dependent manner Fig. . The uprU2OS cells were mainly used in this study because they have been widely utilised in the analysis of DNA repair and signalling due to their intact DNA damage response compared with that of other cancer cell lines. Thus, these findings suggest that the upregulation of PD-L1 occurs in living cancer cells after IR. In contrast, we did not observe an obvious PD-L1 upregulation in primary fibroblast cells (48BR) after DNA damage in the time range of our analysis . Strikingly, IR-, CPT-, Etp- or APH-dependent induction of PD-L1 expression was significantly suppressed by ATM inhibition, suggesting that PD-L1 expression is upregulated in response to DSB-dependent damage signalling . Notably, IR-induced PD-L1 upregulation was substantially suppressed by ATR or Chk1 inhibition  also induce DSBs but indirectly via DNA replication. ATM, which is the most important signal transducer, serves as a sensor of DSBsing Fig. . The MRNSBs Fig. . After Iion Fig. . The PD-ion Fig. . Furtherion Fig. . The uprion Fig. . Taken tThe findings above prompted us to identify the factors regulating PD-L1 expression in DSB repair and signalling. To identify DSB repair factors regulating PD-L1 upregulation after IR, we carried out a screen using an siRNA library targeting DSB repair and signalling . Consistent with the data in the first siRNA screen, depletion of BRCA2 by using another siRNA targeting BRCA2 substantially increased PD-L1 expression after IR Fig. . In a cllls Fig. . The enhlls Fig. . Furtherlls Fig. ; similarlls Fig. . In addi38. The Ku70/80 complex has two major roles in DSB repair. One is the recruitment of NHEJ factors to initiate the downstream part of the repair process. The other is to protect DNA ends from unscheduled digestion by DNA nucleases40. Since DSB-dependent PD-L1 expression requires Chk1 activity, which is activated by ATR on single-stranded DNA (ssDNA)-replication protein A (RPA), we hypothesised that the greater PD-L1 upregulation after IR in Ku80-depleted cells may be due to Chk1 overactivation following an increase in DNA end resection. To address this issue, we examined resection activity and Chk1 signal by monitoring the phosphorylation of RPA Ser4/8 (a resection marker) and Chk1 Ser345, respectively. Greater RPA/Chk1 phosphorylation was observed in Ku80-depleted cells after IR, suggesting that Ku80 depletion enhances DNA end resection and Chk1 activation  in U2OS and H1299 cells . We found that PD-L1 in tumours  with BRCA2, PALB2 or Ku70/80 mutations showed higher expression than in tumours without such mutations  showed that there was no significant difference in PD-L1 expression in TNBC between BRCA1 carriers and noncarriers49. In our screen, BRCA1-depleted cells did not show a significant increase in IR-induced PD-L1 expression compared with that in control cells. These paradoxical results can be explained by our model that Chk1 activation via DNA end resection is a key process in DSB-induced PD-L1 expression. Both BRCA1 and BRCA2 are required for HR, however, BRCA1 and BRCA2 have distinct roles in HR. BRCA1 promotes DNA end resection by relieving the barrier posed by 53BP1 in HR. Therefore, BRCA1 deficiency attenuates the DNA damage signal after IR unless the 53BP1 pathway is functioning normally51. In particular, the ATR/Chk1 signal is not effectively activated if the resection is impaired in BRCA1-defective cells. Notably, the loss of 53BP1, or the loss of RIF1 or REV7, rescues DNA end resection and HR in BRCA1-defective cells54. Disruption of the 53BP1 pathway, which rescues BRCA1 defect, was observed in subsets of sporadic TNBC. Notably, ~70% of BRCA-associated TNBC cases were found to revert to HR via an unknown mechanism. Therefore, the BRCA1 gene status alone may not be simply correlated with PD-L1 expression. On the other hand, BRCA2 is dispensable for resection, but is required for RAD51 loading following the formation of RPA on resected DNA ends. The protein switch from RPA to RAD51 is promoted by BRCA2, and the recruitment of BRCA2 on chromatin requires PALB2. Therefore, the defect of BRCA2 causes the stalling of HR repair at the step of RAD51 loading, but sustains activation of Chk1 at unrepaired DSBs. This effect was confirmed when BRCA2-depleted cells were treated with PARPi. PARP inhibition causes replication fork stalling, which requires BRCA2 to resolve and repair DNA. Notably, the addition of a Chk1 inhibitor cancelled the PARPi-dependent PD-L1 upregulation in BRCA2-depleted cells. Hence, we propose that a defect of DSB repair does not simply influence PD-L1 upregulation; however, the Chk1 activity plays a central role in the upregulation of PD-L1 after DSBs.A recent cohort study reported associations of CD3+ TILs and BRCA1/2 mutation status with survival38. In addition to this role, the Ku complex plays a significant role in protecting DSB ends. To remove the Ku complex from DSB ends safely without excessive resection, the initiation of resection is highly regulated by MRE11/CtIP endonucleases44. However, the lack of the Ku complex causes an increase in DSB end resection, which requires EXO1/BLM40. This increase in resection results in Chk1 activation. We found that depletion of the Ku complex caused high level of PD-L1 expression even without DNA damage. The greater endogenous PD-L1 expression (without DNA damage) in Ku80-depleted cells is likely due to the accumulation of replication-associated damage because the Ku complex also binds to replication-associated DSBs55. In addition to the upregulation of endogenous PD-L1 expression, we found a substantial increase in PD-L1 by Ku depletion after IR or Etp. An experiment using a Chk1 inhibitor clearly revealed that DSB-induced upregulation of PD-L1 in Ku-depleted cells is mediated by Chk1 following the excessive resection, supporting the notion that DSB-induced PD-L1 is regulated in resection-Chk1 pathway. Throughout the present study, we noticed that the magnitude of PD-L1 upregulation between mRNA and protein levels was not always perfectly matched. Because PD-L1 is controlled by post-translational modifications, such as ubiquitination56, and also multiple ubiquitin ligases and deubiquitinating enzymes are activated following DSBs57, the PD-L1 levels may be fine-tuned by the modifications depending on the cellular situation after DNA damage. Further studies are required to reveal the post-translational regulation for controlling PD-L1 expression after DNA damage. In an investigation of clinical specimens, reduction of Ku70/80 expression was observed in samples with the progression of melanoma58. In addition, BRCA2 mutations are enriched in melanomas responsive to anti-PD-1. Thus, downregulation of either Ku or BRCA2 or both may affect the efficacy of PD-1 therapy through PD-L1 upregulation by IR, and also via neoantigen pathway.Ku70/80 is a heterodimer protein that binds to DSB ends and promotes the recruitment of NHEJ proteins11. In addition, a meta-analysis reported that PD-L1 expression on cancer cells is one of the promising candidates as a selection marker17. A phase III clinical trial that involved only patients who had non-small-cell lung cancer with PD-L1 expression on at least 50% of tumour cells showed that pembrolizumab was associated with significantly longer progression-free and overall survival than chemotherapy12. These findings suggest that PD-1 therapy is effective in patients showing high PD-L1 expression. In the TCGA analysis, we showed a correlation between PD-L1 expression and mutations in the BRCA2, PALB2 and Ku70/80 genes. This data set analysis may not represent a direct correlation between PD-L1 expression and DSB signalling because the enhancement might largely be due to the high expression of neoantigens. However, because the statistical significance of the enhancement of neoantigens is less than that of the enhancement of PD-L1, we speculate that DNA damage signalling may partly contribute to the increase in PD-L1 expression in tumours when DNA repair and signalling are downregulated, particularly in patients who are treated with radio/chemotherapy.In previous studies, PD-1 therapy was shown to achieve remarkable clinical responses in patients with several types of cancer; however, ~20–40% of patients still remained poorly responsiveTaken together, in the present study, we showed that the depletion of BRCA2 or Ku80 upregulates PD-L1 expression in the DSB signal cascade. Our siRNA screen showed that the downregulation of DSB repair genes is not always associated with PD-L1 upregulation. Notably, we demonstrated that factors regulating Chk1 activity are important. Thus, BRCA2, PALB2 and Ku70/80 identified in this study could be good markers for predicting the efficacy of combined PD-1 and radio/chemotherapy, although a combined therapy may be sufficiently effective regardless of the gene status. In conclusion, we propose that understanding the signalling pathway underlying the response to DSB induction by radio/chemotherapy, which leads to PD-L1 upregulation, is of high importance to develop prognostic molecular markers and PD-1 blockade cancer immunotherapy. The factors could be pharmaceutical targets to influence the therapeutic efficacy when combined PD-1 and radio/chemotherapy is administered. These open questions will be investigated in future work.59. PARP inhibitor (PARPi)  was also added at 10 μM, and the medium was refreshed with 10 μM PARPi every 48 h until the indicated time point.U2OS, H1299 and DU145 cells were obtained from the American Type Culture Collection (ATCC). Cancer cells were cultured in Eagle’s Minimum Essential Medium (EMEM) with 10% foetal calf serum (FCS). X-ray irradiation was performed at 100 kVp and 20 mA with copper (0.5 mm)–aluminium (1.0 mm) filters . Dose rate was set at 0.5 Gy per min. Etp (Sigma-Aldrich), a topoisomerase II inhibitor, was added at 100 or 500 nM, or CPT (Wako), a topoisomerase I inhibitor, was added at 50 nM, and cells were incubated until harvesting. Etp directly causes DSBs, whereas CPT causes replication-dependent DSBs in S phase cells. APH (Wako), a replicative polymerase inhibitor, was added at 500 nM, and cells were incubated until harvesting. In addition, 10 μM ATM inhibitor (ATMi) , 10 μM ATR inhibitor (ATRi) , Chk1 inhibitor  was added at 30 min prior to DNA damage induction. A previous study has shown that 100 nM UCN-01 specifically inhibits Chk1 but not Chk2siRNA transfection was performed using HiPerFect (Qiagen). siRNA was added to suspended cells after trypsinisation. After 24 h, cells were re-transfected with siRNA in suspended cells after trypsinisation. Cells were incubated for 24 h after the second transfection before DNA damage induction. The sequences of the siRNA oligonucleotides used in this part of the study are listed in Supplementary Table 44. The uncropped versions of immunoblotting are shown in Supplementary Fig. i microscope by using NIS-Elements D imaging software with a 40 × objective via a DS-Qi2 camera. The mean signal intensity of PD-L1 in DAPI-negative areas was measured by ImageJ v1.48. Cells positive for PI, representing dead cells, were excluded from this analysis. Similar results were obtained in at least two independent experiments. The antibodies used are listed in Supplementary Table Immunoblotting was performed by using whole cell lysate−ΔΔCt method. qPCR settings were as follows: initial denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. The primers and probes used for the qPCR are listed below:Total RNA was extracted from cells using NucleoSpin RNA (MACHEREY-NAGEL) at the indicated time points after DNA damage induction. PrimeScript RT Reagent Kit  (TaKaRa) was used to reverse-transcribe cDNA from total RNA, in accordance with the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using StepOnePlus (Life Technologies). Reactions (20 μl each) were prepared in duplicate in MicroAmp Fast Optical 96-Well Reaction Plate (Applied Biosystems). Each reaction contained 0.5 μM of each primer, 0.2 μM probe, 10 μl of Taqman Universal PCR Master Mix (Applied Biosystems) and cDNA as a template. The expression levels were normalised to GAPDH and calculated using the 2PD-L1 forward: 5′-GGAGATTAGATCCTGAGGAAAACCA-3′PD-L1 reverse: 5′-AACGGAAGATGAATGTCAGTGCTA-3′PD-L1 probe: 5′-AGATGGCTCCCAGAATTACCAAGTGAGTCC-3′PD-L2 forward: 5′-GCTTCACCAGATAGCAGCTTTATTC-3′PD-L2 reverse: 5′-CTCCAAGGTTCACATGACTTCCA-3′PD-L2 probe: 5′-CATTCCAGGGTCACATTGCTGCCATGCT-3′GAPDH forward: 5′-CTCCTCTGACTTCAACAGCGA-3′GAPDH reverse: 5′-CCAAATTCGTTGTCATACCAGGA-3′GAPDH probe: 5′-ATGCCAGCCCCAGCGTCAAAGGT-3′Cancer cells were incubated for 48 h after exposure to 10 Gy X-rays or PAPRi with or without inhibitors and then harvested for flow cytometry analysis. Adherent cells were harvested by shake-off in 1 mM EDTA-PBS without trypsinisation. Harvested cells were washed with ice-cold 1 mM EDTA-PBS and then stained with anti-PD-L1 antibodies for 20 min on ice. Dead cells detected by PI (Sigma-Aldrich) were excluded in the analysis. Flow cytometry analysis was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The MFI (PD-L1–isotype) is calculated as: the MFI (PD-L1) is subtracted by the MFI (isotype control).http://tcia.at/). The studies for analysis were chosen based on the following two conditions: the number of cases with wild-type PD-L1  was more than 300 and the number of cases with mutations in each set of genes was more than 1% of the total N. Samples with or without mutations, including indel and point mutations, in BRCA2, PALB2, Ku70/80 or any of these four genes were compared using the Mann–Whitney U-test. Box plots were created using the BoxPlotR tool60. The samples that had the RPKM value = 0 were omitted from the log plots. The results of analyses of other tumours are shown in Supplementary Table The normalised RNA sequence and mutation status data provided by The Cancer Genome Atlas (TCGA) project were downloaded from the Genomic Data Commons Data Portal. The neoantigen data were obtained from The Cancer Immunome Atlas  upon reasonable request.Supplementary InformationPeer Review File