EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50–60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation  increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10–32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of  Recent advances in immune checkpoint therapies are rapidly changing the clinical applications of cancer therapies . ProgramSTAT3  was amplified from genomic DNA purified from NCI-H1373 cells by PCR with PrimeSTAR GXL DNA Polymerase (Takara) and the following primer set: forward primer, 5′-ACCTGAGCTCGCTAGCAACATGACTCACCTGAGGACAA-3′; and reverse primer, 5′-TATCCTCGAGGCTAGCTCCATCCCAAAGAAAGGGTGTAG-3′. The PCR product was subcloned upstream of the luciferase gene of pGL4.17 basic luciferase plasmid (Promega) with the In-Fusion HD Cloning Kit (Takara) (PDL-P).The candidate promoter region  plasmid (Promega), which encodes Renilla luciferase driven by a thymidine kinase promoter, were co-transfected with FuGENE6 (Promega) at a FuGENE (μL) to DNA (μg) ratio of 6. Two hours later, DMSO or U0126 (20 μM) was added. Twenty-four hours after the transfection, the firefly and Renilla luciferase activities were determined with the Dual-Glo Luciferase Assay System (Promega) and an infinite M200 (TECAN) multiplate reader. After subtracting the background activity, each firefly luciferase activity was normalized using Renilla luciferase activity to determine the transfection efficiency.NCI-H1373 cells were dispensed to Nunclon Delta Surface, a 96-well white-bottomed plate  the day before transfection. Upon transfection, the cell density reached approximately 50%. One hundred nanograms of firefly luciferase plasmid  and 100 ng of pGL4.74 [5′-TCACATTTCAAGCAGGATGACTAAA-3′ and reverse primer, 5′-TGACTCACAGCCACTCTTCCA-3′. The amount of immunoprecipitated AP-1-responsive element was determined using a standard curve constructed from the serial dilution of input DNA, and fold enrichment was calculated using the cJun/isotype control value.ChIP was conducted for cJun in NCI-H1373 cells. Immunoprecipitation of the predicted AP-1-responsive element was conducted using the ChIP-IT Express Enzymatic Kit (Active Motif) and cJun mAb  or isotype control mAb , followed by qRT-PCR with SYBR Green Gene Expression Assays and the following primer set: forward primer, PD-L1-high and -low lung cancer cell lines using CCLE Analysis Tools: Differential Expression and Gene Set Enrichment Analysis (GSEA) .Supervised cluster analysis and GSEA were conducted between the t test was used for comparison of the two groups. Data are expressed as mean ± standard deviation (SD). The correlation between MFI and RQ of PD-L1 expression was evaluated by Pearson’s correlation test. Chi-square test was used for comparing the frequency of the two groups, and a p-value of less than 0.05 was regarded as significant.Unpaired Student’s PD-L1 mRNA levels of a human lung cancer cell line panel as the relative quantity (RQ) to that of BEAS-2B, a human bronchial cell line  and PD-L1-low (PD-L1 RQ < 1) groups (PD-L1-high mRNA (R > 2.7) cell lines expressed a substantial amount of PD-L1 on their surfaces suggests that post-transcriptional regulation is not a dominant mechanism regulating PD-L1 levels.We determined ell line  and disc) groups . MFI of ) groups . The cor) groups . AlthougEGFR-mutant and five KRAS-mutant cell lines, the histological characteristics of which resulted six of them being classified as adenocarcinomas and one as a large cell carcinoma, while the PD-L1-low group contained only two KRAS-mutant adenocarcinoma cell lines  reside outside known functional domains or potential phosphorylation sites, the functional significance of these mutations cannot be specified. The frequency of either EGFR or KRAS mutations in the PD-L1-high group was significantly higher than that in the PD-L1-low group (p = 0.00056). We obtained the same result about mutational status of EGFR or KRAS by COSMIC database. We inferred that the downstream signals of EGFR or KRAS might regulate PD-L1 expression.We obtained information about the mutational status of major driver oncogenes or a tumor suppressor gene in the 20 human lung cancer cell lines from the CCLE. The PD-L1-high group contained two ll lines . BecauseKRAS-mutant lung adenocarcinoma cell lines  were evaluated for PD-L1 expression 24 h after treatment with the MEK inhibitor U0126. Flow cytometry analysis revealed a 50–60% decrease of surface PD-L1 expression with U0126 compared with that with DMSO , miR-200b expression in two (NCI-H358 and NCI-H441), and miR-200c expression in two (NCI-H1373 and NCI-H358) cell lines, suggesting that the MAPK signal suppresses some miRNAs, counteracting the post-transcriptional suppression of PD-L1 by miRNAs in some cell lines. However, the increase in miRNA expression with U0126 was generally weak (9–37%) and inconsistent among the cell lines. So we cannot conclude that miRNA is universally regulated by MAPK signaling.To examine the possible post-transcriptional regulation of PD-L1 under an MAPK signal,  cancers , were qureatment . U0126 tEGFR mutation, in the EBC-1 lung squamous cell carcinoma cell line without an EGFR/KRAS/ALK mutation, and in the NCI-H460 lung large cell carcinoma with a KRAS (Q61H) mutation  than in the U0126 experiments (50–60%)  , which m(50–60%) , residuaor STAT3 .PD-L1 promoter fragment (−2403 to +153 from TSS) contains the binding sites for transcription factors except AP-1. A candidate AP-1 binding site was found at chr9:5455433–5455446 according to GRCh37/hg19 in the first intron of the PD-L1 gene by ChIP-seq data from UCSC Genome Browser  and the PD-L1 promoter plus a candidate AP-1 enhancer (PDL-P+E) vectors were constructed  compared with that with U0126, and PD-L1 expression was not affected in one cell line, indicating that the regulatory mechanism of PD-L1 by STAT3 signaling may not occur as widely as MAPK signaling.Next, we evaluated the contribution of signaling pathways other than MAPK signaling downstream of A levels . The degKRAS, was also evaluated. LY294002, a PI3K inhibitor, did not decrease PD-L1 expression in any of the three KRAS-mutant lung adenocarcinoma cell lines , motility (VAV1 and RAC2), cell proliferation (ANXA3 and IFI16), apoptosis (TNFAIP8 and SH3KBP1), cell signaling , ubiquitination (FBXL13), a ligand for NKG2D (RAET1E), or metabolism (CDA) than PD-L1-low cell lines , the functional significance of these differences is questionable. Next, we compared the expression of RAC2 and CDA mRNAs between DMSO and U0126 treatments in the three KRAS-mutant lung adenocarcinoma cell lines  regulating these molecules seems to be, at least in part, MAPK signaling.GSEA showed that two pathways, actin cytoskeleton regulation by Rho GTPases and integrin-mediated cell adhesion, significantly correlated to the ll lines . Both suEGFR [EML4-ALK [NPM-ALK [EGFR mutation [EML4-ALK fusion [KRAS mutation also contributes to enhanced PD-L1 expression. Ota et al. showed that an MEK inhibitor (U0126) and a PI3K inhibitor (LY294002), but not a STAT3 inhibitor (S3I–201), decrease PD-L1 expression in EGFR-mutant and EML4-ALK-positive lung cancer cells, as determined by qRT-PCR and flow cytometry [KRAS-mutant adenocarcinoma cell line. Contrary to the previous report mentioned above, our analysis showed the partial contribution of STAT3, but not PI3K signaling, to PD-L1 expression. The different mutational status may lead to different signaling pathways for PD-L1 expression.The mechanisms of ectopic PD-L1 expression in human cancers have been studied in lung cancers –13, melaEGFR ,11, EML4[NPM-ALK , with ac[NPM-ALK –8. It ha[NPM-ALK ,17–19, S[NPM-ALK , or PI3K[NPM-ALK  are relemutation ,11 and EPD-L1-high cell lines included three squamous cell carcinomas without any known oncogenic driver mutations and one large cell carcinoma with a KRAS (Q61H) mutation  without AP-1 sites  in BEAS-2B does not induce PD-L1 expression [KRAS is insufficient for PD-L1 expression. Skoulidis et al. recently reported three subsets  of KRAS-mutant lung adenocarcinoma cell lines according to the co-occurring genetic events as determined by an integrative analysis of genomic, transcriptomic, and proteomic data [KRAS-mutant lung adenocarcinoma cell lines may reflect their heterogeneity.Two n levels . Akbay epression , suggestmic data . These sRAC2 and CDA as genes with significantly higher expression in PD-L1-high lung cancer cell lines. The expression of these genes as well as PD-L1 was dependent on MAPK signaling, suggesting that MAPK signaling constitutes a common pathway for an immune checkpoint molecule, and other functional molecules regulating cell motility, proliferation, or pyrimidine metabolism. GSEA identified that the pathways of the actin cytoskeleton regulation by Rho GTPases and integrin-mediated cell adhesion are significantly related to PD-L1-high lung cancer cell lines. These signatures may be related to the enhanced cellular motility or invasive ability and are reminiscent of the findings in a previous report by Chen et al. describing the correlation between PD-L1 expression and epithelial—mesenchymal transition (EMT) [KRAS-mutant lung adenocarcinoma cell lines, which was also dependent on MAPK signaling (unpublished data). Although the functional significance of these molecules requires further investigation, we speculate that MAPK signaling regulates gene expression related to EMT and immune evasion. Furthermore, we found CCL17 (TARC) expression was higher, while CXCL3 expression was lower in PD-L1-high cell lines than in PD-L1-low cell lines, respectively (We identified on (EMT) . We alsoon (EMT) –30, in Kectively . CCL17 iKRAS-mutant lung adenocarcinoma cell lines. We also demonstrated the MAPK-dependent transcriptional activity of AP-1, as well as the binding of cJUN to AP-1, in the PD-L1 gene. MAPK signaling also regulates PD-L1 in squamous and large-cell lung carcinoma cell lines. PD-L1 is correlated with the expression of other functional genes (RAC2 and CDA) and to pathways related to the cytoskeleton and cell adhesion, suggesting possible implications for EMT.In summary, this study showed that PD-L1 expression is regulated by MAPK and partially by STAT3 signaling in S1 FigPD-L1-high cell lines (RQ > 2.7 in PD-L1-low cell lines (RQ < 1). Empty and shaded histograms indicate isotype control and PD-L1, respectively. BEAS-2B, a normal human bronchial cell line, is shown as a reference.A, Ten (PDF)Click here for additional data file.S2 FigKRAS-mutant lung adenocarcinoma cell lines showed inconsistent increases in the expression of miR-200a, miR-200b, and miR-200c with U0126 . Each miRNA level was normalized by miR-16  and DMSO-treated cells (reference). Data are the mean of three independent experiments.qRT-PCR (ΔΔCt method) of (PDF)Click here for additional data file.S3 FigEGFR mutation ; (B) EBC-1, a squamous cell carcinoma cell line; and (C) NCI-H460, a large cell carcinoma cell line. Data are the mean of three independent experiments.(A) NCI-H1975, an adenocarcinoma cell line harboring (PDF)Click here for additional data file.S4 FigA, Surface protein (relative MFI) (upper) and mRNA levels (RQ) (lower) of PD-L1. Data are the mean of three independent experiments. B, Immunoblot shows KRAS knockdown and decreased ERK phosphorylation. One representative result of two or three independent experiments.(PDF)Click here for additional data file.S5 FigCCL17 expression is higher, while CXCL3 expression is lower in PD-L1-high than in PD-L1-low lung cancer cell lines.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Flow cytometry was used to examine the expression of PD-L1 on ALDH+ MMICs. To determine the role of PD-L1 in MMICs self-renewal, we cultured melanoma cells with anti-PD-L1 and measured tumorsphere formation and apoptosis. In addition, the effects of anti-PD-L1 on tumorigenicity and residual ALDH+ MMICs in tumors were evaluated in vivo. We demonstrated that melanoma cell-intrinsic PD-L1 was expressed in ALDH+ MMICs. Blocking PD-L1 in melanoma cell lines impaired tumorsphere formation and induced the apoptosis of sphere cells. In addition, blocking PD-L1 inhibited tumor growth in vivo. We observed residual ALDH+ MMICs within the tumor. The results showed that blocking PD-L1 also significantly decreased the residual ALDH+ MMICs in the tumors. In conclusion, these results suggest a new mechanism underlying melanoma progression and PD-L1-targeted therapy, which is distinct from the immunomodulatory actions of PD-L1.Recent studies have indicated that therapeutic antibodies targeting PD-L1 show remarkable efficacy in clinical trials in multiple tumors and that a melanoma cell-intrinsic PD-1: PD-L1 axis promotes tumor growth. However, few studies have shown tumor-intrinsic PD-L1 effects in malignant melanoma initiating cells (MMICs). Here, we aim to determine the possible regulatory effects of PD-L1 on MMICs. The ALDEFLUOR kit was used to identify ALDH Metastatic melanoma is an extraordinarily challenging cancer, with a 16% 5-year survival rate, and it responds poorly to most standard chemotherapies , 3. For Recently, it has been established that programmed death-1 (PD-1) is a prominent checkpoint receptor that, upon binding its ligands PD-L1 or PD-L2, dampens T effector functions by inhibiting signaling downstream of the T cell receptor . PD-L2 i+ tumor-initiating cells [In contrast to the immunosuppressive effect of PD-L1, it is also known to contribute to the promotion of tumor cell growth and downregulation of quiescent cells , 9. Furtng cells . PD-L1 ang cells . These rμg/mL streptomycin (Gibco). Cells were cultured at 37°C in 95% humidity and 5% CO2 atmosphere. All cell lines were routinely screened for mycoplasma contamination.B16-F0 and B16-F1 melanoma cell lines were maintained in RPMI 1640 (Gibco) containing 10% fetal bovine serum , 100 U/ml penicillin (Gibco), and 100 ∗106/ml cells were suspended in Aldefluor Assay Buffer (AAB) and incubated with 5 μL ALDH substrate (BAAA) for 45 min. 5 μL diethylaminobenzaldehyde (DEAB) was added to a separate sample containing BAAA for an ALDH-inhibited control. Then, samples were washed and resuspended in AAB. Fluorescence-activated cell gates were established using the inhibited control, DEAB, with the fluorescein isothiocyanate (FITC) channel with excitation and emission wavelengths of approximately 495 nm and 521 nm, respectively. To evaluate the expression of PD-L1 in ALDH+ cells, PD-L1 antibody  and immunoglobulin G (IgG) isotype-matched control (GeneTex) containing BAAA were added to the cells separately. All samples were incubated for 30 minutes at 4°C. Following incubation, the material was centrifuged, and pellets were resuspended with 500 μl assay buffer prior to data acquisition. Flow cytometry analysis was performed on a BD Biosciences FACSCanto, and data analysis was conducted using CellQuest Pro (B&D Biosciences).The ALDEFLUOR kit  was used to identify the stem/progenitor cells that expressed high levels of the aldehyde dehydrogenase (ALDH) . Brieflyμg/mL heparin (Sigma-Aldrich), 4 mg/mL bovine serum albumin (Sigma-Aldrich), 20 pg/mL insulin (Sigma-Aldrich), N2 supplement (Invitrogen), supplemented with 10 ng/mL bFGF , and 20 ng/mL EGF (Peprotech), as previously described [μg anti-PD-L1  or control rat immunoglobulin G (IgG). Tumorspheres were observed under microscope 14 days later. Individual spheres with diameters larger than 100 μm from each replicate well were visualized and counted with an inverted microscope.The B16-F0 and B16-F1 melanoma cells were plated as single cells in ultralow attachment six-well plates  and cultured in RPMI 1640 containing 6 mg/mL glucose (Sigma-Aldrich), 1 mg/mL NaHCO3 (Sigma-Aldrich), 5 mM HEPES (Sigma-Aldrich), 4 escribed . On the Cells were double-stained with FITC-annexin V and PI according to the manufacturer's instructions (Annexin-V FITC/propidium iodide (PI) Apoptosis Detection Kit; BD Pharmingen). Analysis was performed by flow cytometry. Early apoptotic cells were stained with Annexin-V alone, whereas necrotic and late apoptotic cells were stained with both Annexin-V and PI.5 B16-F0 or 5 × 105 B16-F1 melanoma cells. Then, 100 μg anti-PD-L1 or control rat IgG was administered intraperitoneally 3, 6, and 9 days following melanoma cell inoculation. All animals were randomly assigned to two groups of 5 mice each. Tumor size was monitored every two days. All surgical procedures and care given to the animals were in accordance with institutional guidelines.Adult SPF male C57BL/6 mice were implanted subcutaneously on the right flank with either 5 × 10t-test was used to determine significant differences. P values < 0.05 were considered statistically significant.All data were reported as the mean ± standard error. Statistical analysis was performed using GraphPad Prism 5.0 Software (San Diego). A two-tailed paired +/ALDH+ subpopulations from these two cell lines. As shown in + cells were identified in melanoma cell lines by flow cytometry with the ALDEFLUOR kit. Cells were then incubated for 30 min with mouse monoclonal antibodies specific for PD-L1. The analysis of the percentage of PD-L1+ALDH+ cells was gated by ALDH+ cells. We found that approximately 10% to 18% of the cultured murine B16-F0 cells and B16-F1 cells were ALDH+. Approximately, 5% of the ALDH+ cells were PD-L1+/ALDH+. These data suggest that PD-L1 may be involved in regulating MMICs.Previous studies have described the isolation of MMICs from mice using ALDEFLUOR/ALDH as a marker , 15. To To determine whether PD-L1 can mediate MMIC self-renewal, we cultured melanoma cell lines with anti-PD-L1. The results showed that anti-PD-L1 significantly inhibited tumorsphere formation in B16-F0 and B16-F1 melanoma cells compared to the control groups . Cancer Tumorsphere formation has been reported as a measure of the presence of MMICs in enriched cell populations. We further explored the effects of anti-PD-L1 on apoptosis in melanoma tumorspheres. The data illustrated that anti-PD-L1 induced significant apoptosis in melanoma tumorspheres . Anti-PDμg anti-PD-L1 or control rat IgG 3, 6, and 9 days following melanoma cell injection. Other studies have reported that anti-PD-L1 significantly suppressed tumor growth compared with PBS-injected animals in two animal models. Anti-PD-L1 promoted tumor rejection in 50% of B16-F0 melanoma challenged mice (P = 0.031) and 50% of B16-F1 melanoma challenged mice  and 1.4-fold residual ALDH+ MMICs in the B16-F0 animal model (P = 0.045). These results suggest that one mechanism for the anti-tumor effects of anti-PD-L1 is related to its ability to suppress the tumorigenicity capacity of MMICs.Mice were challenged with melanoma cells (B16-F0 and B16-F1) and treated with 100 + MMICs and induced tumorsphere formation. PD-L1 further inhibited the apoptosis of MMIC-enriched cells. Blockage of PD-L1 directly inhibited tumorigenesis in vivo and significantly decreased the residual percentage of MMICs. These results may indicate that melanoma cell-intrinsic PD-L1 promotes self-renewal and the tumorigenic capacity of MMICs.Previous studies have shown that PD-L1 expression is a common phenomenon in immunotherapy-naive melanomas –19. FurtTraditionally, PD-1 ligands have been expressed in tumor cells, leading to T-cell exhaustion and tumor cell evading the immune response, which was thought to require its receptor interaction . Accordi+ MMICs. Glioma stem cells expressed lower levels of the PD-L1 than those of differentiated glioma cells, which contributed to the higher sensitivity of glioma stem cells to the cytotoxicity of the IL-2-activated NK cells [+ tumor-initiating cells and inhibits IFN-γ secretion by tumor-infiltrating lymphocytes (TILs) incubated with CD44+ cells [ in vivo, which is in agreement with these published studies.MMICs are known to have high ALDH. Previous studies have successfully used ALDH as a marker to isolate MMICs from mice , 15. OurNK cells . In head4+ cells . These p4+ cells –29. We sIt has been established that the expression of PD-L1 is an indicator of poor prognosis for patients' survival in many cancers, such as pulmonary adenocarcinoma, gastric cancer, colorectal cancer, and esophageal cancer –33. In cTaken together, our results suggest that melanoma-PD-L1 can enhance tumorigenesis by maintaining the stem cell properties in MMICs. Future studies are needed to elucidate the underlying cellular and molecular mechanisms, which will be helpful to maximize its clinical benefits.


The surface and interior of the support has a porous architecture formed by RGO/SiO2 sandwich structure. The sandwich structure is like a brick wall coated with a graphene layer of concreted as skeleton which could withstand external pressures and endow the structure with higher support stabilities. After loading the Z-N catalyst, the active components anchor on the surface and internal pores of the supports. When the ethylene molecules meet the active centers, the molecular chains grow from the surface and internal catalytic sites in a regular and well-organized way. And the process of the nascent molecular chains filled in the sandwich structure polymerization could ensure the graphene disperse uniformly in the polymer matrix. Compared with traditional methods, the porous spherical graphene support of this strategy has far more advantages and could maintain an intrinsic graphene performance in the nanocomposites.Reduced graphene oxide/SiO Graphene, a monolayer of carbon atoms which has a tight packing of honeycomb lattice and serves as the basic building block of graphitic materials, has recently become one of the most appealing stars in material science . GraphenGenerally, solution mixing is one of the ideal ways in making polymer/graphene nanocomposites if the polymer can be readily dissolved in common organic solvent. At the same time, melt mixing is also adoptable for polymer/graphene nanocomposite fabrication, which in fact is applicable to not only those solvent-resolvable polar polymers but also nonpolar polymers like polyolefin. Although the two methods have been applied to many projects, there are some drawbacks as well, for example, during the process of composite fabrication, the polymer/graphite nanocomposites could not obtain well-dispersed graphene sheets .As graphene is stable and lacks functional groups, it can hardly react with the target polymer or has a good dispersion in most common solvents. It is of great interest to implement nanofiller in order to optimize the material properties. And GO platelets have been utilized as a catalyst support for the heterogeneous in situ polymerization of polyolefin, which reacts with a catalytic component by surface functional groups of GO . This shSummarizing the existing reports on the preparation of polymer/graphene nanocomposites, in situ polymerization shows well-dispersed few-layer GO platelets, but composites with nonpolar polymer category like PE or PP are not able to reduce GO to graphene thermally. Catalytic components have to react with GO by surface functional groups and then obtain more severely complicated surface functional groups, which throw the graphene into disorder. The existence of defects and the disordered structure reduced some of the most important properties of graphene such as thermal conductivity, electrical conductivity, mechanical strength, and carrier mobility –29. Rese2 nanocomposite as support for Z-N catalyst with a novel 3D porous architecture was synthesized by the self-assembly and spray freeze-drying method. It can reach a molecular-scale homogeneity and has minimal agglomeration, and also, the size of the nanocomposite could attain 20–50 μm. In addition, we use the new support synthesis Bi-supported Ziegler-Natta catalysts of the TiCl4(C4H9MgCl(BuMgCl)/RGO-SiO2) system, and the uniform and dispersed, replicable morphology of supports, a roundish graphene/ultrahigh molecular weight polyethylene(UHMWPE) composite, has been synthesized. These properties are beneficial to continuous operation of the preparation and production equipment. Also to date, no reports have the synthesis of granuliform or roundish graphene/UHMWPE composites, which is a reappearance of the spherical morphology of the graphene-based support in the polymerization process. The nanocomposites synthesized by a Ziegler-Natta catalyst system using the micron spherical RGO-SiO2 supports have high-efficient graphene and reduce many unnecessary defects, which are usually generated from in situ polymerization.To address the challenges above, we have been focusing on preparing a kind of high-efficient graphene-based support to reduce unnecessary defect generated from in situ polymerization as much as possible. The graphene-based catalyst supports act as the framework for loading catalyst in its large surface and internal pores. Meanwhile, the spherical graphene support takes the role of nanometer reinforcing filler and conductive agent. During the process of olefin polymerization, the graphene-based catalyst cracks and all the graphene layers disperse in the polyolefin matrix uniformly. Here, a unique micron spherical reduced graphene oxide(RGO)/SiO2, RGO-SiO2, BuMgCl/GO-SiO2, and BuMgCl/RGO-SiO2 were obtained with a Horiba Jobin Yvon LABRAM-HR800 with a wavelength range of 0–4000 cm−1. The Fourier transform infrared spectra (FT-IR) of the samples were recorded using a Nicolet NEXUS 670 FT-IR spectrophotometer, and the sample and KBr were pressed to form a tablet. The transmission electron microscopy (TEM) images were taken by the Hitachi H-600 microscope. Thermogravimetric analysis (TGA) for the samples was performed on a Perkin–Elmer diamond thermal analyzer from room temperature to 600 °C, using a heating rate of 10 °C/min with N2 as the sample purge gas. The fracture surface of the composites and semi-quantitative composition were analyzed by scanning electron microscopy energy dispersive X-ray detector . The melting point of polymers was measured by differential scanning calorimetry (DSC) at a heating rate of 5 °C/min on a Perkin-Elmer Pyris diamond thermal analyzer under the nitrogen flow rate of 30 ml/min from 25 to 400 °C. The measurement of the molecular weight was carried out on a Ubbelohde viscometer according to the Mark-Houwink equation: [η] = 1.1 × 10−4 Mη0.8.X-ray diffraction (XRD) patterns were recorded by a Rigaku D/Max-2400 diffractometer using Cu Kα radiation. Raman spectra of the GO, BuMgCl/GO, RGO, GO-SiO2 were monitored using TEM. Figure 2; the adjacent nanoparticles of SiO2 are easily agglomerated due to high surface energy in water. The decoration of SiO2 in the GO-SiO2 hybrid is revealed by the TEM image of Fig. 2 line up together and are wrapped in GO sheets tightly, forming a sandwich structure. The detailed pathway of building a sandwich structure from GO sheets and SiO2 is shown in Fig. Morphological changes starting from GO to GO-SiO2 hybrid  catalyst, which shows that the morphology is destroyed again based on the original sphere. But the support has little impact; therefore, the overall morphology is preserved. EDX spectroscopy of the TiCl4/(BuMgCl/RGO-SiO2) catalyst in a selected area shown in Fig. 2 carrier in addition to C, O, Si, Mg, and Cl  and the D band at 1342–1357 cm−1 (associated with the order/disorder of sample). Obviously, the D band for the starting materials is relatively intense comparing to its G band, which is typical for small stacks in the defective /[Ti] ratio used during the polymerization. Generally, higher [TEA]/[Ti] resulted in higher activity. DSC studied the thermal properties of the obtained UHMWPE/GO composites. A consistent polymer melting temperature near 142 °C is reassuring. The PE/GO composites with graphene loading of 0.28 and 0.5 wt%  were obtained. The samples were subjected to viscosity measurement to determine their molecular weights (Mη). Accompanied by an increase in the amount of TEA, the viscosity-average molecular weights of the UHMWPE matrixes were changed from 3.1 × 106 to 2.0 × 106 g/mol, which are UHMWPE requirement. Morphological characterization of UHMWPE/RGO-SiO2 is shown in Fig. 2 composite granules have uniform size, good dispersion, and regular sphere-like shape. The average diameter of the composites has also been calculated. The statistics of particle distribution is shown in Fig. 2 supports. As the average particle size is 150 μm, which is three times of the support size, and the proportion of 120~180 μm particles is more than 70% of all statistics, that was very similar to the size distribution of GO-SiO2 supports, and also, the morphology of composites repeats. The detailed pathway of morphology repetition is shown in Fig. 2, which could explain the molecular chain growth on the surface and surroundings of catalyst.Propylene polymerization was performed with the TiCl2 spherical support, is synthesized through the reaction of thermal reduction with GO-SiO2 spherical composite at 700 °C in an argon atmosphere to remove residual oxygen groups internally from GO-SiO2 sandwich structure in the RGO-SiO2 sandwich structure and activate silica at the same time. Then, it reacts with BuMgCl in tetrahydrofuran (THF) to synthesize Bi support, followed by an excessive TiCl4 treatment to generate Mg/Ti catalyst component; a portion of component formation happens directly on the surface of support and other parts pass through the porous architecture into the internal and interact with silica nanoparticles that scattered around the sandwich structure. Then, the supported catalyst is engaged in ethylene polymerization. When ethylene met the active center, the molecular chains of the support surface grow and crystallize quickly in the surrounding of the catalyst, along with the vertical direction of spherical tangent. And the molecular chains of the internal catalytic sites also begin to grow. They filled around the RGO-SiO2 sandwich structure and has no interaction with graphene; therefore, graphene can disperse uniformly. With the further growing of molecular chains, the volume of polymer granules grows, and it becomes even more difficult to repeat the morphology completely. This is the reason why the nanocomposites are not near-perfect spheres in the SEM shown in Fig. 2 sandwich structure.Polymer morphology repetition based on a reaction scheme is illustrated in Fig. −4 S/cm; with decreasing GO content, it was 4.62 × 10−5 S/cm. Comparing to the method that used dispersed graphene nanosheet directly, this method will cost less content of graphene in forming the nanocomposites to achieve the same conductivity. The reason is that in the former method, many catalytic groups of the supported catalyst was contained on the graphene surface, then destructing the original conjugated system and deteriorating its conductivity. However, after the spherical supports are synthesized, the reaction of a Grignard reagent BuMgCl with GO-SiO2 in THF occurs on the support surface, which will not damage the internal graphene structure, and a conductive network is formed as the internal graphene scattered in the polymer while the polymerized samples are mixed and compressed to films.The electrical conductivity of melt-compressed UHMWPE nanocomposites was measured using the four-point method on cylindrical plates with 20 mm diameter and a height of 2 mm. At 0.5 wt% GO content, the electrical conductivity was 6.46 × 102 nanocomposite as a new support for Ziegler-Natta catalyst with a 3D spherical porous architecture was synthesized. We attempted to prepare the support by reducing the unnecessary defects generated during the process of in situ polymerization. The supports act as catalyst frame, reinforcing nanofillers and conductive agents. The catalytic component and active sites are uniform and well-distributed in the surface and internal pores of the supports after using the novel support synthesis Bi-supported Ziegler-Natta catalysts of TiCl4(BuMgCl/RGO-SiO2) system. And the morphologies of the polyethylene composites replicate the morphologies of the supports, then spherical UHMWPE/graphene composites have been synthesized, which is beneficial and critical to operate the continuous reparation and production equipment in the industry. In addition, these RGO-SiO2 nanocomposites with porous architecture also have potential applications in many other fields, such as being catalysts, energy storage materials, nanoelectronics, and photo-electronic devices.In this study, a unique micron RGO/SiO


Recent advances in optical technologies such as multi-photon microscopy and optogenetics have revolutionized our ability to record and manipulate neuronal activity. Combining optical techniques with electrical recordings is of critical importance to connect the large body of neuroscience knowledge obtained from animal models to human studies mainly relying on electrophysiological recordings of brain-scale activity. However, integration of optical modalities with electrical recordings is challenging due to generation of light-induced artifacts. Here we report a transparent graphene microelectrode technology that eliminates light-induced artifacts to enable crosstalk-free integration of 2-photon microscopy, optogenetic stimulation, and cortical recordings in the same in vivo experiment. We achieve fabrication of crack- and residue-free graphene electrode surfaces yielding high optical transmittance for 2-photon imaging down to ~ 1 mm below the cortical surface. Transparent graphene microelectrode technology offers a practical pathway to investigate neuronal activity over multiple spatial scales extending from single neurons to large neuronal populations. Optical imaging and manipulation technologies cannot be easily integrated with electrical recordings due to generation of light-induced artifacts. Here the authors report the optimization of transparent graphene microelectrode fabrication to achieve artifact-free electrical recordings along with deep 2-photon imaging in vivo. Multi-photon imaging has enabled cellular-resolution imaging of neural populations in animal models, whereas optogenetics (OGs) has been widely employed for selective control of neural activity and casual manipulation of specific neural circuits. High temporal resolution of electrophysiological recordings is critical to complement optical techniques for investigating fast dynamics of neural activity toward understanding functions of neural circuits. Furthermore, vast majority of early neuroscience research and clinical human studies rely on electrophysiological recordings of brain-scale activity. Combining optical techniques with electrical recordings can bridge the classical neuroscience knowledge obtained from animal models to human studies based on electrophysiological techniques. To that end, new tools allowing simultaneous measurements of multiple optical and electrical parameters are essential. Previous reports have shown proof-of-concept acquisition of local field potentials (LFPs) by graphene-based electrodes during simultaneous single-photon fluorescence imaging in vitro2, or OG photostimulation in vivo3. However, especially recordings during in vivo OG photostimulation have significantly suffered from light-induced artifacts, not showing any noticeable advantage over conventional platinum electrodes3. The problem of light-induced artifacts needs to be addressed, as it constitutes a major obstacle particularly for the adaptation of transparent graphene electrode technology in chronic in vivo studies.Multimodal integration of sensing and manipulation technologies allows comprehensive investigation of brain function across spatiotemporal scales7, due to increased laser power. Here, in the present study, we demonstrate that by careful design of key steps in the fabrication process for transparent graphene electrode, the light-induced artifact problem can be mitigated and virtually artifact-free LFP recordings can be achieved within operating light intensities. High optical transmittance of graphene supports simultaneous 2-photon imaging down to > 1 mm directly beneath the transparent microelectrodes. For the first time, we show that transparent graphene electrodes can be employed for crosstalk-free integration of three different modalities, 2-photon imaging, OGs, and electrical recordings of cortical potentials at the same time in the same experiment. We combine LFP recordings from the cortical surface, with simultaneous (i) 2-photon imaging of neuronal and vascular structure down to ~ 1 mm below the cortical surface, (ii) 2-photon imaging of neuronal calcium activity, (iii) single-photon OG photoactivation, (iv) 2-photon imaging of arteriolar vasodilation, and (v) large-scale optical imaging of hemodynamic responses. Crosstalk-free integration of various in vivo optical imaging and stimulation methods with graphene electrode recordings proves that transparent graphene technology is a versatile platform applicable to numerous different experimental settings. In cases where depth-resolved electrical recordings are not required, optically transparent graphene technology allows seamless integration with depth-resolved optical imaging and stimulation circumventing the need for inserting invasive probes into brain tissue.Light-induced artifacts generated by photovoltaic (Becquerel effect) and photothermal effects appear as transients or oscillations in recordings and can interfere with LFP or spike recordings, depending on the frequency and duration of the light stimulus. The amplitudes of those artifacts are particularly higher for deep 2-photon imaging and OG stimulation3 and poly(methyl methacrylate) (PMMA) scaffold transfer2 are prone to formation of cracks on the monolayer graphene sheets resulting in low yield for large-area planar arrays. In addition, copper etching used in these processes leaves residues impeding the process of PMMA removal8. As a result, the graphene layers may lose their structural integrity during annealing. Therefore, we adopted an alternative “bubbling transfer” process9 for crack-free transfer of graphene sheets onto the polymer substrate (see Methods). To protect the graphene during photoresist stripping we used polymethylglutarimide (PMGI)-based bilayer lithography. Compared with other photoresists, PMGI leaves fewer residues on the graphene surface after resist removal. Photoresist removal is also critical to reduce crack formation in subsequent fabrication steps. To this end, we developed a four-step stripping/cleaning protocol for extensive cleaning of the graphene surface leading to a robust reduction of the average electrode impedance in the array  graphene transfer, (2) photoresist removal from the graphene surface, and (3) graphene surface cleaning from organic residues. We first optimized the process of graphene transfer from growth to target substrate. This process is the most critical, yet most sensitive step of the fabrication. Previously used techniques, such as wet transfer grapheneWe fabricated 4-by-4 arrays of 100 µm × 100 µm square graphene electrodes separated by 300 µm (edge-to-edge) Fig. . FabricaFollowing the fabrication, we characterized each graphene microelectrode array in 0.01 M phosphate-buffered saline (PBS) with electrochemical impedance spectroscopy and cyclic voltammetry (CV) Fig. . Figure X and Y axis) and a 1.75-fold increase in apparent bead size along the Z axis size was observed for beads below microarray substrate and graphene electrode.To examine to which extent the presence of the graphene microarray affects the resolution of images acquired with 2-photon laser scanning microscopy, we placed microarray devices in a phantom sample above mounting medium containing 1 µm fluorescent polystyrene beads at a low concentration  at 470 nm)  plane parallel to the cortical surface, with individual images spacing 3 μm along the depth (Z) axis. Figure 10. EGFP was excited at its peak resonance of 905 nm and detected with a 490–560 nm bandpass filter  into cortical layer II/III of the primary somatosensory cortex and applied Sulforhodamine 101 (SR101) to label astrocytesdow Fig. . To stimdow Fig. . In a moude Fig. , which i16. Experiments were performed in Thy1-ChR2 transgenic mice expressing the OG actuator channelrhodopsin-2 (ChR2) in layer V pyramidal neurons , which is considerably higher than the power typically used for OG photostimulation17.Crosstalk-free integration of optical imaging, OGs, and electrophysiological recordings is critical for investigating neural activity on a circuit or population level, whereas, at the same time, examining the causal role of individual neurons or groups of neurons in circuit function. We investigated the performance of graphene electrode arrays in studies involving both 2-photon imaging and single-photon OG photostimulation. To this end, we performed electrical recordings with graphene arrays and OG stimulation via a cylinder-shaped blue (473 nm) laser beam delivered through the microscope objective with ~ 210 μm in diameter (FWHM); this stimulation protocol has been used in a recent study from one of our laboratoriesons Fig. . We illuons Fig. ; the strons Fig. . We used16. To exclude that vascular responses were elicited through tissue heating (see ref. 18), especially due to light absorption by the microarray, we applied the same OG stimulus in a ChR2-negative mouse and did not observe any stimulus-induced changes of arteriole diameters, which were measured close to the cortical surface , deoxyhemoglobin (Hb), and total hemoglobin (HbT) to detect stimulus-induced cortical hemodynamic activity , while imaging through a graphene surface array for simultaneous LFP recordings Fig. . For speTransparent graphene array technology provides a viable complementary alternative to needle arrays for multimodal measurements/manipulations within the penetration depth of multiphoton microscopy. In cases where depth-resolved electrical recordings are not required, optically transparent graphene surface arrays allow seamless integration with depth-resolved optical imaging modalities while circumventing the need to insert invasive probes into brain tissue. This technology is also well-suited for neurovascular and neurometabolic studies providing a “gold standard” neuronal correlate for optical measurements of vascular, hemodynamic, and metabolic activity.20. Several recent publications have demonstrated successful combination of electrode array recordings with OG photostimulation through integration of optical waveguides or incorporation of LEDs along penetrating electrode shanks26. As a further step in this direction, ongoing efforts are focused on the addition of photodetectors along the electrode shanks that would provide optical imaging capability alongside with electrical recordings and OG photoactivation21. This strategy is particularly attractive for deep brain studies targeting beyond the penetration limits of multiphoton microscopy28. Within these limits, however, simultaneous electrical recordings and optical imaging/manipulation can be achieved by engineering optically transparent electrode arrays31. To this end, in the present study, we introduced a new transparent graphene microelectrode array and demonstrated its unique capabilities for integration with 2-photon imaging and single-photon OG photostimulation.Advancements in measurement technology play a critical role in neuroscience enabling scientific inquiry and powering discovery. This is also the goal of the ongoing BRAIN Initiative32.In this study, transparent graphene array technology has been significantly advanced beyond previously reported transparent electrode demonstrations by incorporating a new graphene-friendly fabrication process. This new fabrication process avoids any crack formation in the transfer process proving 95–100% yield for the electrode arrays. Such techniques can as well be directly employed to fabricate high-density, large-area transparent arrays to monitor brain-scale cortical activity in large animal models. The fabrication process is also complementary metal-oxide-semiconductor (CMOS)-compatible, so that graphene arrays can be directly integrated with amplifying and multiplexing circuits on the same chip34. Thus, combination of surface LFP recordings with 2-photon imaging of neuronal calcium would allow investigation of spiking activity in specific neurons (resolved with 2-photon imaging) in the context of circuit behavior, e.g., “up” and “down” states36.With this technology, we demonstrate simultaneous mapping of surface LFP and high-resolution 2-photon imaging of neuronal calcium transients. LFP signals recorded from the cortical surface reflect flow of currents along the vertically aligned pyramidal cells’ apical dendrites, largely produced by synaptic inputs. Therefore, these signals provide a measure of circuit activity1. Likewise, simultaneously acquired surface LFP and 2-photon measurements of single-vessel dilation as well as mesoscopic measurements of cortical hemodynamics would help neurovascular studies aiming towards the physiological underpinning of human noninvasive functional magnetic resonance imaging (fMRI) signals37.This combination of measurements is also of relevance for studies aiming to bridge neuronal activity across scales and ultimately connect the large body of classical neuroscience knowledge, obtained from research in model systems , to human noninvasive electro-/magnetoencephalography measurements39, glial function40, vascular dynamics37, immune response41, energy consumption42, and more. With the current trends to move away from anesthesia and towards longitudinal imaging in awake behaving mice43, incorporation of graphene surface arrays within a chronic cranial window implant44 would facilitate the adoption of our technology. To this end, ongoing efforts in our laboratories are focused on engineering such double-duty implants providing a transparent optical window and capability of space-resolved surface LFP measurements. For multiphoton imaging, further improvement of image quality can be achieved by addition of adaptive optics to correct for distortions of the excitation wavefront due to mismatched refractive indices between graphene/PET substrate and cortical tissue45.With rapid advancement in multiphoton microscopy and a growing arsenal of synthetic and genetically encoded optical probes, we envision that recordings with optically transparent graphene electrode arrays will be combined with a wide range of microscopic physiological parameters related to neuronal activity31 by combination with charge-coupled device (CCD)-based imaging of vascular/hemodynamic signals, can provide an intermediate step in bridging brain phenomena across scales. Scaled up to cover a larger cortical area, our technology may also be very informative for studies of large-scale, multi-area neuronal activity/connectivity46, as well as for neuronal underpinning of large-scale spontaneous hemodynamic oscillations and their correlation across different regions of the cerebral cortex47. As a nonmagnetic material, graphene array technology can also be utilized to develop fMRI-compatible implantable arrays.Compatibility with large FOV imaging, exemplified in previous studiesTo conclude, we envision that our transparent graphene-based electrode array technology will find application in multiple areas, advancing our understanding of how microscopic neuronal activity at the cellular scale translates into macroscopic activity of large neuronal populations and providing a neuronal correlate for optical measurement of vascular, hemodynamic, or metabolic activity.9 was adopted to protect graphene from cracking during the process. Therefore, 300 nm-thick 495 PMMA A4 was spin-coated on the graphene/copper bilayer structure. This PMMA/graphene/copper tri-layer was then connected to the cathode of a 20 V DC power supply, whereas the anode was submerged in 0.05 M NaOH in deionized water. As the trilayer was gently and gradually immersed into the NaOH solution, hydrogen gas bubbles formed in between the graphene and copper layer, and exfoliated PMMA/graphene from the copper foil. The graphene layer needed to be thoroughly cleansed by floating on the surface of deionized water for three times before it was placed onto the gold pads on the flexible substrate.The bubbling transfer methodGraphene electrode impedance and susceptibility toward light-induced artifacts are highly dependent on photoresist residue. Therefore, we developed a four-step stripping/cleaning protocol to remove organic residues from the graphene surface that was crucial to achieve arrays with low impedance at high yield. After oxygen plasma etching, the sample was soaked in AZ 1-Methyl-2-pyrrolidon and Remover PG to remove AZ1512 and PMGI, respectively. Another acetone bath was used to remove Remover PG residue. All three rinses were conducted at room temperature with gentle agitation. The last cleaning step included ten cycles of isopropanol/deionized water rinse.Using the new graphene transfer and photoresist removal methods, we developed the following fabrication process  in the surgical microscope light source to avoid OG photostimulation during installation of the cortical window. Mice were anesthetized with isoflurane during surgical procedures . A catheter was inserted into the femoral artery. A metal holding bar was glued to the temporal bone for immobilization of the head during imaging. A ~ 4 × 4 mm cranial window was centered on the whisker area of primary somatosensory cortex; overlying skull contralateral to the holding bar was exposed and dura mater removed.All experimental procedures were performed in accordance with the guidelines established by the UCSD Institutional Animal Care and Use Committee. We used 9 adult mice of either sex including three Thy1-ChR2-YFP , two GAD67-GFP13 within the whisker area of the somatosensory cortex. SR101  in ACSF was applied topically for ~ 2 min to label astrocytes12, providing a contrast in tissue that was used for visual assessment of potential damage due to experimental procedures. Excess dye was washed away with ACSF.In calcium imaging experiments, 50 μg calcium indicator Oregon Green 488 BAPTA-1 AM ester  was first dissolved in 4 μl of 20% Pluronic F-127 in dimethyl sulfoxide ; 80 μl of ACSF were added to the OGB1 solution to yield a final concentration of 0.5 mM OGB1. OGB1 solution was pressure-microinjected into the cortical tissueThe graphene array was placed on the cortical surface, a drop of 0.7% (w/v) agarose  in ACSF was applied on top of the array, and the exposure was covered with a rectangular glass coverslip and sealed with dental acrylic. To avoid herniation of the exposed brain due to excessive intracranial pressure, the dura mater over the IVth cerebral ventricle was punctured, thus allowing drainage of CSF. After the exposure was closed, the drainage hole was sealed with agarose.–1 h–1, C0128, Sigma or 100459, MP Biochemicals). Mice were paralyzed with pancuronium bromide 48, and ventilated (~ 110 min−1) with 100% O2. α-chloralose, pancuronium bromide, or 5% dextrose in saline were supplied through the femoral line every 30 min for the duration of data acquisition. Expired CO2 was measured continuously using a micro-capnometer . Heart rate, blood pressure, and body temperature were monitored continuously. Blood gas was analyzed to cross-validate micro-capnometer measurements. Respiration was adjusted to achieve a PaCO2 between 30 and 40 mm Hg and pH between 7.35 and 7.45.After closing the exposure, mice were left to rest under 1% isoflurane for 45 min, which minimized leakage of drugs onto the exposed cortical tissue through cut dural blood vessels. Then, isoflurane was discontinued and anesthesia was maintained with α-chloralose  or 2 MDa dextran-conjugated Alexa Fluor 680 were injected through the femoral line (50–100 μl of 5% (w/v) solution in PBS). To prepare 2 MDa Alexa Fluor 680-dextran, Alexa Fluor 680 NHS ester  was conjugated to 2 MDa amino dextran  using a custom conjugation protocol that can be found on the Devor lab academic website http://nil.ucsd.edu/ under “Resources”.11. FITC, EGFP, and Alexa Fluor 680 were imaged using cooled GaAsP PMT tube detectors . SR101 was imaged using a multialkali PMT .Images were obtained using an Ultima 2-photon laser scanning microscopy system from Bruker Fluorescence Microscopy (formerly Prairie Technologies) equipped with an Ultra II femtosecond Ti:Sapphire laser (Coherent) tuned to 905  nm for imaging of EGFP and 950 nm for imaging of FITC. For penetration deeper than ~ 600 μm, an optical parametric oscillator  pumped by the same Ti:Sapphire laser was tuned to 1280 nm. The OPO was used in conjunction with intravascular administration Alexa Fluor 680-dextranIn experiments involving OG stimulation, the main dichroic mirror contained a 460–480 nm notch (Chroma ZT470/561/NIR TPC). An additional filter that blocks wavelengths in between 458 and 473 nm (Chroma ZET458-473/561/568/NIR M) was added in front of the PMT block. Nevertheless, residual bleed-through of the 473 nm light prevented us from using GaAsP PMT detectors. Therefore, in these experiments, FITC was imaged using a multialkali PMT. For imaging Alexa Fluor 680-dextran, custom-made filters (Chroma) were used; the main dichroic mirror contained a 440–480 nm notch and allowed light transmission from 780 to 1400 nm, the corresponding filter in front of the PMT blocks light between 440 and 480 nm, and above 750 nm.We used 4×  = 0.28) or 5×  objectives to obtain low-resolution images of the exposure. Olympus 20×  water-immersion objectives were used for high-resolution imaging. The laser beam diameter was adjusted to overfill the back aperture. Arteriolar diameter measurements were performed in a “free-hand” line-scan mode with a scan rate of 25–50 Hz. The scan resolution was 0.5 μm or less. Calcium imaging was performed in frame-scan mode at ~ 10 Hz per frame.Electrical stimulation was delivered to the whisker pad contralateral to the cortical exposure through a pair of thin needles inserted under the skin using 300 µs, 1 mA electrical pulses. For calcium imaging, 50 trials with 5 s interstimulus interval (ISI) were delivered at each measurement location. For mesoscopic imaging of cortical hemodynamics, a train of six electrical pulses  were delivered at a rate of 3 Hz with an ISI of 30 s.The stimulation device  was triggered using a separate PC that also acquired timing signals for data acquisition  and physiological readings using a National Instruments IO DAQ interface (PCI-6229) controlled by custom-written software in MATLAB . The timing of electrophysiological acquisition as well as each optical frame/line relative to the stimulus onset was determined during data analysis based on acquired triggering signals.16. The beam was directed to defined locations in the sample using a dedicated set of galvanometer mirrors. The duration of the light pulse was controlled by a dedicated shutter and synchronized with imaging. Laser power was measured by directing the entire beam into the sensor of a broadband power meter . The beam diameter was measured with a CCD-based laser beam profiler .In 2-photon experiments, OG stimulation was delivered though the objective using a 473 nm cylinder-shaped CW laser beam ~ 210 μm in diameter (FWHM)The electrophysiological data were recorded by the RHD2000 amplifier board and the RHD2000 evaluation system from Intan Technologies. The sampling rate was 10 kHz. The DC offset was removed by the built-in filters of the system. Data were analyzed in MATLAB using custom-written software. After import and conversion into MATLAB files, trigger signals were detected, artifacts from electrical stimulation were removed by linear interpolation, if necessary, and the sampling rate was reduced to 1 kHz for trial average and further processing. For normalization, data recorded 50 ms before stimulus delivery were averaged and used as baseline value.40. For analysis of calcium imaging data, neuronal cell bodies were segmented from composite red (SR101) and green (OGB1) images. For individual regions of interest (ROIs), the calcium signal per frame was calculated as average OGB1 fluorescence of all pixels within the ROI. This calculation was repeated for each frame in the time series to generate a single-ROI time-course. All pixels outside astrocytic and vascular ROIs and neuronal cell bodies were specified as neuropil. Vessel diameters were measured using continuous line scans across the vessel that form a space-time image when stacked sequentially. Diameters are extracted from profile changes resulting from expansion or contraction of the intravascular lumen, which is labeled with FITC- or Alexa Fluor 680-dextran.Data were analyzed in MATLAB using custom-written software as described in49. Briefly, six different bandpass filters were placed on a six-position filter wheel (Thorlabs), which was mounted on a DC motor. The center wavelength of the filters ranged from 560 to 610 nm with 10 nm intervals. Light from a tungsten-halogen light source  was directed through the filter wheel, which was coupled to a 12 mm fiber bundle. Images were acquired with a cooled 16 bit CCD camera . Image acquisition was triggered at ~ 18 Hz by individual filters in the filter wheel passing through an optic sensor. The image set at each wavelength was averaged across trials and averaged data was converted to changes in HbO and Hb at each pixel using the modified Beer Lambert relationship as detailed in ref. 49. Baseline concentrations of 60 and 40 μM were assumed for HbO and Hb, respectively51.Spectral imaging of blood oxygenation was performed as previously described in52.The graphene microarray device was fixed at its connecting wire to a 22 × 22 mm glass coverslip with UV-curable optical adhesive (Norland 61). Four 3 mm coverslips serving as spacers were fixed at each corner of the coverslip. One micrometer fluorescent polystyrene beads  were diluted to a final dilution of 1:10,000 in ProLong Gold mounting medium . A drop of bead-containing mounting medium was added onto a glass slide and the coverslip with array was placed on top. Two-photon imaging was performed after curing of the mounting medium at an excitation wavelength of 800 nm using a 20× UMPlanFI (NA = 0.5) water-immersion objective. Data were analyzed in ImageJ using the MetroloJ pluginImproved array performance in vitro has been shown in four independent manufacturing processes with three arrays in each batch.Animal experiments were designed as proof-of-principle experiments to demonstrate graphene microarray performance in vivo; statistical comparison of experimental groups is therefore not applicable. Structural imaging in GAD67-GFP was performed in two independent experiments; calcium imaging with OGB was performed in four independent experiments; experiments involving OG stimulation were performed in three Thy1-ChR2 animals and one wild-type animal; and macroscopic hemodynamic imaging was performed in one animal.The data that support the findings of this study are available from the corresponding authors upon reasonable request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


These power densities are the highest values report for milliliter‐scale MFCs with similar chamber size (25 mL) under the similar measurement conditions. The 3D N‐GA electrode shows great promise for improving the power generation of MFC devices.A 3D nitrogen‐doped graphene aerogel (N‐GA) as an anode material for microbial fuel cells (MFCs) is reported. Electron microscopy images reveal that the N‐GA possesses hierarchical porous structure that allows efficient diffusion of both bacterial cells and electron mediators in the interior space of 3D electrode, and thus, the colonization of bacterial communities. Electrochemical impedance spectroscopic measurements further show that nitrogen doping considerably reduces the charge transfer resistance and internal resistance of GA, which helps to enhance the MFC power density. Importantly, the dual‐chamber milliliter‐scale MFC with N‐GA anode yields an outstanding volumetric power density of 225 ± 12 W m Based on the success of CNT‐based electrode, a variety of 3D graphene‐based materials, such as chitosan/vacuum‐stripped graphene scaffold,−2 −3 .2 g−1) than other carbon materials  such as carbon cloths, carbon black powder, graphite powder as well as some porous metal substrates, e.g., nickel foams (≈0.1 m2 g−1). Because of the aforementioned advantages, graphene aerogels have already been demonstrated as promising electrode materials for supercapacitors,et al. demonstrated an MFC with l‐cysteine tailored porous graphene aerogel as bioanode.−2  but limited volumetric power density of 0.68 W m−3 .3D carbon‐based electrodes are promising candidates for addressing the aforementioned challenges.−3 based on the volume of the anode chamber , which is substantially higher than those of MFCs with conventional carbon cloth (denoted as CC) bioanode  as well as reduced graphene oxide coated nickel foam (denoted as rGO‐Ni) bioanode In this work, we demonstrated the first time the MFC with porous nitrogen‐doped graphene aerogel (denoted as N‐GA) as bioanode. The MFC achieved a low internal resistance of 55.4 Ω and a remarkably high volumetric power density of 225 ± 12 W m22.1FigureShewanella oneidensis)‐enriched solution into the interior portion of the 3D network, which can increase the surface area available for bacterial colonization.Figure2 g−1, which was substantially larger than that of CC (5.3 m2 g−1)2 g−1, Figure S2, Supporting Information). Moreover, we also determined the electrochemically accessible surface area (ECSA) of CC, rGO‐Ni, and N‐GA . Again, the N‐GA possesses the highest ECSA among the three electrodes, consistent with the BET results. The large surface area and hierarchical porous structure containing micro‐, meso‐, and macropores offer large accessible surface for colonization of bacteria and interaction with electron mediators.Brunauer‐Emmett‐Teller (BET) test was carried out to further characterize the porous structure of N‐GA. As shown in 2.2Figure−3 normalized to the total volume of the anodic chamber , which is higher than the CC MFC (17 ± 1 W m−3) and rGO‐Ni MFC (83 ± 2 W m−3). To our knowledge, these volumetric densities are among the highest values of all milliliter‐scale MFCs with similar chamber size and the same catholyte (potassium ferricyanide) (Table2) for N‐GA MFC is 1990.8 ± 106.1 mW m−2, which is much higher than the values of CC MFC (78.2 ± 2.7 mW m−2) and rGO‐Ni MFC (517.6 ± 8.0 mW m−2). Significantly, the maximum areal power density is also among the state‐of‐the‐art performance and considerably higher than other MFCs with carbon‐based bioanodes, including the MEMS MFC (47 mW m−2),−2)−2).To evaluate the performance of N‐GA, we fabricated an MFC using the N‐GA as anode (denoted as N‐GA MFC). The performance of MFCs using the CC and rGO‐Ni as anodes  was also tested for comparison. Notably, as shown in 2.3Figureρ is the bacterial area density (cells mm−2), N is the number of bacterial cells shown in corresponding SEM images and A, the area shown in corresponding SEM images estimated using the scale bars (μm2). The bacterial areal density on the interior surface of rGO‐Ni and N‐GA is estimated to be 19 102 and 89 779 cells mm−2, respectively. It explains why the MFC with rGO‐Ni or N‐GA as anode achieved considerably higher current and power density than the CC MFC. The better performance of N‐GA MFC relative to rGO‐Ni MFC is believed to be due to two reasons. First, N‐GA has higher surface area than that of rGO‐Ni, providing more sites for bacterial colonization, and thus, enhances bioelectricity generation. Second, the nitrogen‐doping could increase the affinity between N‐GA electrode and bacteria. The improvement of bacterial colonization in the interior surface of N‐GA was ascribed to the formation of surface nitrogen‐containing functional groups  that turned graphene sheets into positively charged.SEM images were collected for bacterial colonized CC, rGO‐Ni, and N‐GA electrodes after testing in MFC for 5 d. In the case of CC, bacterial communities were sparsely distributed on exterior surface of the electrode but hardly observed on the interior surface of CC fibers Figure4a. Sin2.4FigureRΩ) and charge transfer resistance of anode  for the three MFCs were obtained . The rGO‐Ni MFC has the lowest RΩ value due to the presence of highly conductive nickel frameworks , while the other two MFCs have comparable RΩ values. Notably, the N‐GA MFC has the smallest Rct,a (11.05 Ω), which was 67 times and 4.5 times lower than that of CC MFC (748 Ω) and rGO‐Ni MFC (61.45 Ω), respectively. The charge transfer resistance is mainly due to the resistance of extracellular electron mediator diffusion.Rct,a value suggests that the diffusion of electron mediators in N‐GA is most efficient among the three anodes. The results also explain why N‐GA MFC achieved the highest power density since the diffusion of electron mediators is one of the key factors determining power density. In addition to RΩ and Rct,a, diffusion resistance (RD) that mainly accounts for the resistance of bacterial culture diffusion has been also widely used in evaluating the performance of MFCs.RD of 81.14 Ω, which is smaller than that of rGO‐Ni MFC (99.52 Ω) and substantially lower than that of CC MFC (237.1 Ω). This result supports our hypothesis that the porous structure of N‐GA allows efficient diffusion of bacterial culture.To evaluate the effect of nitrogen doping on the electrical conductivity of GA and how it affects the performance of MFC, we conducted electrochemical impedance spectroscopy (EIS) measurements for the three MFCs Figure5. By fI–V curve and cyclic voltammograms collected for graphene aerogel without N‐doping (GA) and N‐GA  also confirmed the enhanced electrical conductivity of N‐GA. As a result, the N‐GA MFC displayed a considerably smaller RΩ (55.4 Ω) than that of GA (96.2 Ω) , which is beneficial for N‐GA MFC to achieve the high power density.Moreover, we also investigated the effect of nitrogen‐doping on internal resistance of MFCs. Previous studies have showed that nitrogen‐doping can substantially increase the electrical conductivity of graphene by lowering the energy gap between the highest occupied molecular orbital and lowest unoccupied molecular orbital.3−3 with a high open‐circuit potential of 0.69 ± 0.01 V. The incorporation of N‐GA bioanode makes our MFC achieved the highest volumetric power density ever reported for milliliter‐scale MFCs under similar testing conditions. This work demonstrates the key factor to improve power density of MFCs is twofolded, the synthesis of 3D electrodes with large surface area as well as the successful utilization of interior surface of substrates. Large surface area is important not only for anode but also cathode, so we believe that the N‐GA could also be a good cathode material . Besides, the facile hydrothermal preparation of N‐GAs can be easily scaled up, and thus opens up tremendous opportunities to further push the power densities of large‐scale MFCs.In this work, we have synthesized the highly conductive and porous nitrogen‐doped graphene aerogel MFCs. The N‐GA MFC achieved an outstanding volumetric power density of 225 ± 12 W m4Preparation of Graphene Oxide Solution: GO solution was prepared via a modified Hummer's method reported elsewhere−1.Synthesis of N‐GA: N‐GAs were prepared by a facile hydrothermal method reported elsewhere.−1 GO precursor solution. Then, the mixture was transferred into a Teflon‐lined reactor and sealed in a steel autoclave. The autoclave was heated in an electric oven at 180 °C for 12 h. The nitrogen‐doped graphene hydrogel was formed in the reactor. The graphene hydrogel was immersed in deionized water overnight to remove impurities. Finally, the N‐GA was obtained by freeze‐drying the graphene hydrogel for 12 h. For comparison, graphene aerogels without N‐doping (denoted as GA) were prepared using the same method except concentrated ammonium hydroxide solution was replaced by the same amount of de‐ionized water for the hydrothermal reaction.Material Characterizations: The morphology of the N‐GA was characterized by a field‐emission scanning electron microscope . The specific surface area of N‐GA was determined by Brunauer–Emmett–Teller (BET) method through nitrogen adsorption–desorption at 77 K. Pore size distribution was examined by a porosimetry analyzer  and analyzed according to the Barett–Joyner–Halenda (BJH) model. The composition of N‐GA was characterized by X‐ray diffraction  and X‐ray photoelectron spectroscopy .The binding energies of XPS peaks were calibrated using the C 1s photoelectron peak at 284.6 eV as the reference. The chemical composition of the nitrogen‐containing functional groups was probed by Fourier‐transform infrared spectrometer . Zeta‐potential measurements were conducted with a Nano ZS Zetasizer  using a He‐Ne laser at a wavelength of 632.8 nm.MFC Setup: A drop of Shewanella oneidensis MR‐1 (ATCC 700550) bacterial culture was inoculated into 50 mL of fresh trypticase soy broth  and incubated under aerobic conditions with continuous shaking at 30 °C for 12 h. Then the bacterial suspension was transferred into the anodic chamber and purged with nitrogen gas for 15 min to remove the dissolved oxygen gas in the solution. 20 mL of 15.6 g potassium ferricyanide (K3[Fe(CN)6]) diluted in 100 × 10−3 M phosphate buffer solution (PBS) was used as catholyte and injected into the cathodic chamber. The two chambers were separated by a piece of cation‐exchange membrane (CEM). The volume of each chamber was 25 mL. Carbon cloth  and N‐GA were used as cathode and bioanode, respectively. The MFC was then connected to an external resistance (500 Ω) and the voltage was recorded by a data acquisition unit (Keithley 2700). Once the voltage dropped to background, fresh TSB medium was injected to the anodic chamber. This process was repeated for three times to inoculate Shewanella oneidensis MR‐1 cells. As comparison, MFCs with a piece of carbon cloth  and a piece of reduced graphene oxide coated nickel foam (rGO‐Ni) as bioanodes were assembled according to the same aforementioned method. The volumes of N‐GA, CC and rGO‐Ni were all ≈7.5 cm3.Electrochemical Measurements: Electrochemical impedance spectroscopy (EIS) was performed in a frequency range from 0.01 to 10000 Hz using a CHI 660D Electrochemical Workstation (CH Instrument). An external voltage of 0.1 V was applied to the MFCs with a perturbation of 10 mV during the test. EIS data were fitted using the ZsimpWin software. To collect the polarization curves, MFCs were connected in series with various external resistors . The current (I) was evaluated according to Ohm's law: U is the potential recorded by the data acquisition unit and R is the resistance of the external resistor. The volumetric power density (Pv) was calculated using V is the volume of the bioanode or the anodic chamber. Amperometric I–t curves were collected in a fed batch model for 5 d at maximum power density. The external resistor connected in series with the CC MFC, rGO‐Ni MFC, and N‐GA MFC has a resistance of 100, 100, and 50 Ω, respectively. The potential was recorded by the data acquisition unit (Keithley 2700). The recorded potential was then converted to current according to the Ohm's law. Electrochemically accessible surface area of each electrode was determined by cyclic voltammetry using 5 × 10−3m potassium ferricyanide aqueous solution containing 0.1 m LiClO4 as the supporting electrolyte.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion. Chronic Hepatitis B Virus (HBV) infection is a liver disease affecting approximately 250 million people globally, resulting in estimates nearing 1 million HBV-related deaths annually , but whiex vivo PD-L1 blockade improved responses to HBV antigens by PBMCs or intrahepatic lymphocytes from some but not all CHB patients [in vitro results are similar to clinical experience with PD-1 or PD-L1 blockade in cancer and Hepatitis C, where therapeutic responses affecting the course of the disease can be profound but are experienced by only a subset of subjects [In contrast to the chronic infection state, immune clearance of HBV is accompanied by polyfunctional, polyspecific T cell responses against viral antigens –12. The patients ,14, withpatients . These isubjects ,22.in vivo models. Preclinical in vivo testing of HBV immunotherapies is crucial to assess the efficacy in clearing the HBV DNA reservoir but also to assess the potential for liver damage resulting from immune cytolysis of infected hepatocytes. The infection of woodchucks with woodchuck hepatitis virus (WHV), a hepadnavirus related to HBV, has long been used as a model for pathogenesis and therapy of HBV (19). The WHV model represents the best available model for testing αPD-L1 immunotherapy as PD-1 and PD-L1 are similar to the human orthologues and their expression can be up-regulated in peripheral blood and liver of chronic WHV-infected animals [in vitro blockade of woodchuck PD-L1 and PD-L2 during WHV antigen stimulation improved virus-specific responses in T cells from a subset of animals [While the above results are promising, the safety and efficacy of PD-L1 blockade for CHB has not been robustly tested with relevant  animals –25. Simi animals . HoweverTo further evaluate the therapeutic potential of PD-L1 blockade in CHB, we developed a monoclonal antibody against woodchuck PD-L1 that blocks the PD-1/PD-L1 interaction. This antibody exhibits properties similar to the human monoclonal αPD-L1 antibody BMS-936559 that has demonstrated efficacy in oncology studies . The antProduction and characterization of antibody wc6D5 will be described elsewhere (manuscript in preparation). Briefly, mice were immunized with purified recombinant woodchuck PD-L1 ectodomain, and hybridoma supernatants were tested for the ability to bind to woodchuck PD-L1, to block the woodchuck PD-1/PD-L1 interaction, and to recognize PD-L1 expressed on woodchuck leukocytes. Supernatants were also selected by virtue of recognizing both human and woodchuck PD-L1. This identified mouse IgG1 MAb 6D5. Antibody 6D5 was then cloned and expressed as a chimeric antibody having the same variable regions but substituting the mouse IgG1 constant regions with woodchuck IgG constant regions, with an engineered D265A mutation included to minimize effector function ,28. Isot® Eu-W1024 Anti-Human IgG (Perkin Elmer) and SureLight™ Allophycocyanin-anti-6His (Perkin Elmer) were diluted in LANCE® Detection Buffer and 5 μL was dispensed per well (Perkin Elmer). The final assay concentration of LANCE® Eu-W1024 Anti-Human IgG and and SureLight™ Allophycocyanin-anti-6His were 1 nM and 20 nM, respectively. The binding assay was incubated at room temperature for 1 hour and fluorescence emission was measured at two wavelength (665 nM and 620 nM) using a Perkin Elmer EnVision 2104 Multi-Plate Reader. The FRET ratio and percent inhibition were calculated as recommended by the manufacturer’s instructions. For PD-1/PD-L1 assays, recombinant human or woodchuck PD-1 ectodomains (corresponding to amino acids 25–167 in human PD-1), each with a C-terminal human IgG epitope tag, were used at a final assay concentration of 20 nM. Recombinant human or woodchuck PD-L1 ectodomains (corresponding to amino acids 18–239 in human PD-L1), each with a C-terminal 6xHis tag, were used at a final assay concentration of 10 nM. For the CD80/PD-L1 assay, human CD80-His  and human PD-L1-Ig (RnD Systems) were used at final assay concentrations of 80 and 10 nM, respectively. For the PD-1/PD-L2 assay, human PD-1-Ig and human PD-L2 (RnD sytems) was used at final assay concentrations of 20 and 2 nM, respectively.All HTRF assays were performed at room temperature using low-volume, 384 well, non-treated, round bottom plates (Corning) in a final assay volume of 10 μL. Binding partners were diluted in HTRF assay buffer consisting of Dulbecco’s PBS  supplemented with 0.1% (w/v) Bovine Serum Albumin (Sigma) and 0.05% (v/v) Tween 20 (Bio-Rad) and 2.5 μL of each was dispensed per well. HTRF detection antibodies, LANCE2 incubator for overnight incubation. The next day, antibody was added followed by the addition of 5 μL/well of PD-1-Ig-PE to cells at a final concentration of 0.5 nM. Sample were incubated for 1 hour at 37 °C for 1 hour, followed by 3 washes in 100 μl of dPBS. Cells were fixed by adding 8% (w/v) formaldehyde (Sigma), 20 μg/mL Hoechst (Thermo Scientific) in PBS for 20 minutes at room temperature. The plates were washed 3 more times in dPBS and data was obtained using a CellInsight NXT High Content Screening Platform IC903000 (Thermo Scientific). The 50% effective concentration (IC50) was calculated using the four-parameter logistic formula y = A+((B-A)/(1+((C/x)^D))), where A and B denote minimal and maximal % inhibition, respectively, C is the EC50, D is hill slope and x represent compound concentration.Recombinant human or woodchuck PD-1-Ig was directly labeled with R-PE using an R-PE conjugation kit (Abcam) according the manufacturer’s instructions. Human embryonic kidney cell expressing the SV40 virus large T antigen (293T) were obtained from the American Type Culture Collection. 293T clones stably expressing human PD-L1 (293T-hPDL1) or woodchuck PD-L1 (293T-wcPDL1) were seeded into PDL-coated 384 well plates  at 2,000 cells per 20 μL of culture media consisting of DMEM (Life Technologies) supplemented with 10% (v/v) FBS and 1X Penicillin/Streptomycin (Life Technologies). Plates were allowed to incubate for 10 minutes at room temperature and then transferred to a 37°C/5% COWoodchucks were purchased from Northeastern Wildlife  or bred at the Roswell Park Cancer Institute. Animals were housed at Northeastern Wildlife, Roswell Park Cancer Institute, or Bristol-Myers Squibb for various studies. When euthanasia was necessary, animals were euthanized by intravenous injection of Beuthanasia-D Special .7 genome copies with subsequent monitoring of plasma anti-WHV core antibody, WHV sAg, and WHV DNA to confirm chronicity, and were used at > 1 year of age. Naturally infected animals were wild-caught and chronic infection status was confirmed by presence of anti-WHV core antibody, WHV sAg, and WHV DNA at stable levels for at least 3 months prior to use. Naive animals did not show evidence of WHV exposure, as indicated by seronegativity for anti-core and anti-sAg antibody, sAg, and WHV DNA. For acute infection studies, adult animals negative for WHV sAg, anti-WHV core antibody, and anti-WHV sAg antibody were used. Animals were inoculated intravenously with 107 genome copies of WHV strain 7 virus to initiate acute infection.Laboratory infected, chronic WHV+ animals were generated by subcutaneous inoculation at ~3 days of age with WHV strain 7 at a quantity of 10Entecavir was dissolved at 0.1 mg/ml in a solution of 50% Phosphate Buffered Saline and 50% Woodchuck Liquid Diet , and administered daily at 1 ml/kg via an oral dosing needle for a dose of 0.1 mg/kg/day. Antibodies were administered by slow intravenous bolus injection at a rate of ~6 ml/minute using 5 mg/ml solutions at doses as indicated in figure legends. In all studies, wc6D5 and wcAnti-KLH were administered in four doses every 3 to 4 days over a 10 day period.5’-CCTCGCAGGAGAAGATCTCAA-3’, WHV-SL-R: 5’- GCAGTTGGCAGATGGAGATTG-3’, and minor groove binding probe WHV-SL-MGB: 5’-6FAM-ACCGCGTCGCAGAC-3’, within the core/polymerase overlapping region of the WHV genome. WHV sAg, anti-core antibody, and anti-sAg antibody were measured as previously described [Circulating viral loads were determined by qPCR as previously described , or alteescribed ,31. WHV 6mm punch biopsies were collected by laparotomy from isoflurane-anesthetized woodchucks, fixed in 10% neutral buffered formalin and paraffin-embedded.Immunohistochemistry analysis was performed on FFPE tissues for antibodies directed against PD-L1 , CD3 , and MAC2 . The sections were manually deparaffinized through several changes of xylene and ethanol to rehydrate followed by antigen retrieval with a pressure cooker  for 30 minutes at 95°C using ER1 solution . The slides were then loaded on an Intellipath automatic system . Endogenous peroxidase was inactivated using Peroxidazed 1  for 5 minutes. PD-L1 (1:100), CD3 (ready to use), and MAC2 (1:1000) were automatically applied to separate slides and incubated for 120, 60, and 60 minutes, respectively. The slides for PD-L1 and CD3 were then incubated with Leica Post Primary and Leica Rabbit HRP-Polymer each for 30 minutes. The slides for MAC2 were incubated with Biocare Rat HRP-Polymer (one step) for 30 minutes. Visualization was facilitated by IP FLX diaminobenzidine (DAB) chromagen. Slides were then counterstained with hematoxylin.Image Analysis To quantitatively assess the expression of PD-L1, CD3, and MAC2, each entire IHC liver section from each animal was scanned at 40X magnification and digitized using Aperio ScanScope & Image Analysis System . The images were then transferred to a HALO image analysis system . To quantify the expression of PD-L1, CD3, and MAC2, the entire liver section was selected, the analysis was performed by using Indica Cyto Nuclear V1.4 algorithm. Briefly, the IHC image was analyzed by color for each stain used. Nuclear stain (blue) and Positive Stain 1 (brown) were assigned to hematoxylin for total nuclear staining and DAB for the PD-L1, CD3, or MAC2 positive staining, respectively. After trained, each specific algorithm for PD-L1, CD3, and MAC2 was assigned. The percentage of Positive 1 (DAB) from each animal was used to compare among the groups.Complete blood counts were analyzed on Idexx Procyte DX or Abaxis HM5 automated analyzers, or were analyzed by the Cornell University Animal Health Diagnostic Center. Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALKP), Gamma Glutamyl Transferase (GGT), Total Bilirubin (TBIL), Glucose (GLUC), Urea Nitrogen (BUN), Creatinine (CREA), Total Protein (TP), Albumin (ALB), Cholesterol (CHOL), Triglycerides (TRIG), Calcium (CA), Inorganic Phosphorus (PHOS), Sodium (NA), Potassium (K), and Chloride (CL) were evaluated on an ADVIA 1800 Automated Chemistry Analyzer , or were analyzed by the Cornell University Animal Health Diagnostic Center.Analysis of PD-1 on total CD3+CD4-negative (CD8) T cells was performed on heparinized whole blood. 100μl of blood was stained with anti-PD-1 MAb 27F1 (3 μg/ml), followed by detection with goat anti-mouse IgG subclass 1 specific-phycoerythrin secondary antibody (Jackson ImmunoResearch). Cells were then washed and lymphocyte populations labelled with woodchuck cross-reactive anti-rat CD3-APC clone 1F4 (BD Biosciences) and anti-human CD4-PERCP clone L200 (BD biosciences), and fixation with Cal-Lyse-Whole blood Lysing Solution (Invitrogen). Flow cytometry was performed on a BD FACSVerse using FACSDiva software and analysis in FlowJo X.0.7.wc6D5 in serum samples was measured by capture ELISA. Serial dilutions of serum from wc6D5-treated animals were incubated on microtiter plates coated with purified recombinant His-tagged woodchuck PD-L1, then plates were washed and wc6D5 detected with Protein G-biotin (Pierce) followed by streptavidin-HRP and detection with TMB substrate and absorbance reading at 450 nm. A standard curve of serum with known wc6D5 concentration was used to convert absorbance to antibody concentration.6 PBMCs in a total volume of 1ml per well and incubated at 37°C, 5% CO2 for 6 days. Following in vitro antigen-specific expansion, cells were resuspended by vigorous pipetting and transferred to a 2 ml 96-well block plate (Costar). Cells were collected by centrifugation at 350 x g for 10 minutes, then resuspended in 450 μl of media with 10% FBS and 15-mer peptide libraries. One-hundred and fifty microliters of cells per condition were transferred to duplicate wells of PVDF 96-well plates coated with anti-woodchuck IFN-γ monoclonal antibody 7H10 at 2 different seeding densities, 1.5e5 and 6e5 cells per well, performed in duplicate. ELISpot plates were incubated for 24 hours at 37°C, 5% CO2, then washed and spots detected with anti-IFN-γ monoclonal antibody 27C11  followed by goat anti-mouse IgG2b specific-HRP conjugate (Jackson Immuno Research) and TMB ELISpot substrate (MabTech). Developed ELISpot plates were scanned and spot forming units quantitated using the ImmunoSpot ELISpot plate counter from CTL.PBMCs were isolated from lithium heparin-anticoagulated blood via standard Ficoll methodology. The final cell pellet was resuspended in CTS OpTimizer T-cell Expansion SFM supplemented with L-Glutamine (Life Technologies) and 10% FBS. Cells were stimulated with overlapping 15-mer peptide libraries  spanning the complete coding sequence of WHV core or sAg  in 48-well plates with 2x10Guide for the Care and Use of Laboratory Animals, the United States Department of Agriculture’s regulations CFR9 1.1–3.142, and in compliance with all state, local, and federal laws, the Animal Welfare Act, and the PHS Policy on the Humane Care and Use of Laboratory Animals, under protocol numbers 106-16WC and 1064W.Animal work was reviewed and approved by the IACUCs at Bristol-Myers Squibb, Northeastern Wildlife and Roswell Park Cancer Center. The studies were conducted following the National Research Council’s in vivo for only short durations of treatment due to the rapid induction of anti-mouse IgG antibodies. Since pharmacodynamic effects of T cell checkpoint blockade may require weeks to become evident [We wished to use the chronic WHV model to evaluate the potential benefit versus risk for treatment of CHB patients with the human monoclonal antibody (HuMAb) αPD-L1, BMS-936559, previously used in oncology studies (17). However, BMS-936559 does not cross-react with woodchuck PD-L1. Therefore, we needed an antibody with reactivity to woodchuck PD-L1, as well as pharmacokinetics (PK) and effector function that would approximate the characteristics of BMS-936559 in humans. We developed a surrogate monoclonal antibody able to bind and functionally block the woodchuck PD-1/PD-L1 interaction with potency similar to that of BMS-936559 for blockade of human PD-1/PD-L1 . This an evident ,22, we gin vivo, we first tested wc6D5 as a monotherapy in viremic animals with established chronic WHV infection. A cohort of 10 laboratory-bred, chronically WHV+ animals was treated with either wc6D5 (n = 6) or vehicle control . Four doses of wc6D5 over 10 days at a 15 mg/kg/dose displayed extended PK with plasma levels exceeding the expected saturating concentration of 1 ng/mL for greater than 60 days from the start of treatment  for 5–12 weeks in treated animals . ETV pluA major concern with any novel immunotherapy for CHB is that induced immune responses could cause unacceptable liver damage in the process of controlling viral infection. However, no marked increase in ALT or AST levels occurred in the three treatment-responding woodchucks during combined ETV plus wc6D5 therapy , or in aWhile αPD-L1 is a promising therapy for chronic infections, significant immune-related side effects can occur ,22. Idenin vivo treatment response  may have limited the duration of PD-L1 blockade and therapeutic effect. Lastly, like many studies with WHV+ woodchucks, only a very small numbers of animals were included in each treatment group, so an infrequent response in a subset of animals may not necessarily have been detected. We expand upon this earlier work by using a well-characterized monoclonal antibody to achieve robust and prolonged PD-L1 blockade, having a sufficient number of animals to detect responses in a minority of treated animals, and characterizing multiple peripheral and hepatic parameters in each study as well as WHV-naïve, lab-infected and naturally-infected WHV+ animals.PD-L1 blockade has been tested in the HBV DNA hydrodynamic injection model in mice. Virus clearance was accelerated when treatment was initiated prior to DNA injection; treatment after establishment of chronicity was not tested . Only onodchucks . In thatSustained antiviral effects were seen with αPD-L1 plus ETV treatment that were not observed with ETV treatment alone. Large reductions in plasma sAg occurred shortly after initiation of PD-L1 blockade in 3 of 11 treated animals. This effect was maintained after cessation of ETV treatment in these same animals. Similar reductions and/or continued suppression also was observed for viral DNA and eAg. In 2 of the responder animals (#7095 and 7802), these benefits continued well after wc6D5 was cleared from circulation compare , S2 Fig,ex vivo rate of response when αPD-L1 is applied to woodchuck PBMCs ex vivo [The response rate observed here with αPD-L1 plus ETV  is similar to the response rate observed in a phase 1 clinical trial of αPD-1 (nivolumab) in HCV-infected subjects (3/20 at 10 mg/kg dose) , the rat ex vivo . In the  ex vivo –47, and  ex vivo ,49. Simi ex vivo –14, and  ex vivo ,16,37.In humans, upregulation of PD-1 or PD-L1 is not observed in all CHB patients, but rather correlates with inflammation and immune-active disease –20,35. DAlthough the reductions in WHV DNA and WHsAg reported here with αPD-L1 therapy are encouraging, they stop short of the ultimate goal of anti-sAg antibody seroconversion. No induction of anti-sAg antibody occurred in any of the chronically infected woodchucks treated with wc6D5, although the study did not have sufficient numbers of animals to detect seroconversion rates similar to those seen with IFN treatment (~10%). Perhaps the study was terminated prior to achieving levels of WHsAg and CCC DNA reduction necessary for seroconversion. Predictably, greater efficacy may also be achieved by using αPD-L1 in combination with other novel therapies including therapeutic vaccines and antivirals  or antig6 copies/mL) is a predictor of ex vivo response to αPD-L1 with human intrahepatic lymphocytes [Meanwhile, it may be possible to identify patient subgroups in which αPD-L1 achieves larger or more consistent therapeutic effects. Low pre-treatment viremia Click here for additional data file.S2 Fig(A) Plasma levels of anti-woodchuck PD-L1 Mab wc6D5 was determined in plasma of treated animals at various times post-infection. Treatment responders are shown in green; animals treated with wc6D5 that did not show an antiviral response are in blue. (B-D) Comparison of wc6D5 PK with viral load and sAg kinetics in treatment responders. Individual graphs are shown for each animal. sAg data are shown at left, viral load data are shown at right. Horizontal axes are at the limit of detection for each parameter: 200 ng/ml for wc6D5, 500 copies/ml for viral load, and 20 ng/ml for sAg.(TIF)Click here for additional data file.S3 FigPBMCs were isolated and tested for responses to WHV core and sAg peptide libraries as described in Materials and Methods. Results for each individual woodchuck are shown. ELISPOT responses are displayed as fold change over unstimulated controls for WHV cAg library (blue bars) and sAg library (red bars). Viral load (green) and sAg (yellow) in the same animal is overlaid on each graph. wc6D5 treatment responder animals 7095, 7254, and 7802.(TIF)Click here for additional data file.S4 FigPBMCs were isolated and tested for responses to WHV core and sAg peptide libraries as described in Materials and Methods. Results for each individual woodchuck are shown. ELISPOT responses are displayed as fold change over unstimulated controls for WHV cAg library (blue bars) and sAg library (red bars). Viral load (green) and sAg (yellow) in the same animal is overlaid on each graph. wc6D5 treatment non-responder animals 6707, 7060, 7800, and 7802.(TIF)Click here for additional data file.S5 FigPBMCs were isolated and tested for responses to WHV core and sAg peptide libraries as described in Materials and Methods. Results for each individual woodchuck are shown. ELISPOT responses are displayed as fold change over unstimulated controls for WHV cAg library (blue bars) and sAg library (red bars). Viral load (green) and sAg (yellow) in the same animal is overlaid on each graph. Antibody wc6D5 treatment non-responder animals 7806, 7808, 7809, 7801.(TIF)Click here for additional data file.S6 FigPBMCs were isolated and tested for responses to WHV core and sAg peptide libraries as described in Materials and Methods. Results for each individual woodchuck are shown. ELISPOT responses are displayed as fold change over unstimulated controls for WHV cAg library (blue bars) and sAg library (red bars). Viral load (green) and sAg (yellow) in the same animal is overlaid on each graph. Antibody isotype-control treated animals 7799, 7811, and 7813.(TIF)Click here for additional data file.S7 FigPBMCs were isolated and tested for responses to WHV core and sAg peptide libraries as described in Materials and Methods. Results for each individual woodchuck are shown. ELISPOT responses are displayed as fold change over unstimulated controls for WHV cAg library (blue bars) and sAg library (red bars). Viral load (green) and sAg (yellow) in the same animal is overlaid on each graph. Antibody isotype-control treated animals 7584 and 7810.(TIF)Click here for additional data file.S8 FigAnimals undergoing acute WHV infection were treated with wc6D5 at 1 mg/kg (blue) or 15 mg/kg (purple), or with isotype control MAb wc6D5 at 15 mg/kg (red). Antibodies were administered in four doses over 10 days, starting on week 7 post-WHV infection. Viral loads for each individual animal in each group are shown.(TIF)Click here for additional data file.S9 FigPlasma levels of anti-woodchuck PD-L1 mAb wc6D5 was determined in plasma of treated animals at various times post-infection. Animals received either 1 mg/kg (blue) or 15 mg/kg (purple) wc6D5 in four doses between days 49 and 59 post infection.(TIF)Click here for additional data file.