The long-lasting impact might be mediated by an enhancement of memory extinction or an impairment of memory reconsolidation. Here, we directly compared the effects of i.p. injections of cannabidiol (10 mg/kg) with those of the NMDA receptor antagonist MK-801 (0.1 mg/kg) and partial agonist D-cycloserine  in order to determine the mnemonic basis of long-term fear reduction. We showed that under conditions of strong fear conditioning, CBD reduced contextual fear memory expression both acutely during the extinction session as well as later at a fear retention test. The latter test reduction was replicated by DCS, but MK-801 instead elevated test freezing. In contrast, when initial conditioning was weaker, CBD and MK-801 had similar effects to increase freezing at the fear retention test relative to vehicle controls, whereas DCS had no observable impact. This pattern of results is consistent with CBD enhancing contextual fear memory extinction when the initial conditioning is strong, but impairing extinction when conditioning is weak. This bidirectional effect of CBD may be related to stress levels induced by conditioning and evoked at retrieval during extinction, rather than the strength of the memory  Cannabis plant and has anxiolytic therapeutic potential. Acute administration of CBD decreases the expression of contextual conditioned fear memories in rats  is the major non-psychotropic constituent of the CBD can also result in long-lasting fear reduction, when combined with either extinction training or brief memory reactivation/destabilization to impair reconsolidation  were housed in quads under a 12 h light cycle (lights on at 0700) in a specialist animal facility. Individually-ventilated cages contained aspen chip bedding and a plexiglass tunnel for environmental enrichment. Rats had free access to food and water other than during behavioral sessions. Experiments took place between 0900 and 1200 in a behavioral laboratory. At the end of the experiment, animals were humanely killed using a rising concentration of CO2. All procedures were approved by the local animal welfare and ethical review board and carried out in accordance with the United Kingdom 1986 Animals (Scientific Procedures) Act, Amendment Regulations 2012 (PPL 70/7662).Drugs were administered intraperitoneally (1 ml/kg) 30 min prior to extinction sessions. CBD  was dissolved in DMSO and diluted to a final vehicle of 20% DMSO in Saline with 0.1% Tween 80 immediately prior to use. MK-801  and D-cycloserine  were dissolved in saline vehicle and stored at −10°C until use. Doses of drugs were taken from published literature showing modulatory effects on fear memory  as previously described (Lee and Hynds 2013), with freezing behavior automatically recorded by Videotracking software . All rats received the same behavioral training, apart from a single difference in the conditioning session.One day prior to conditioning, rats were exposed twice to the conditioning chamber for 10 min on each occasion, with a 60-min ITI. Conditioning consisted of either a 7-min or a 10-min session for weak and strong conditioning, respectively. For both conditions, there was a 5-min pre-shock period, followed by exposure to a sequence of 0.5-mA, 1-s footshocks at 300 s and 341 s . On the next day, rats were given i.p. injections 30 min prior to a 20-min re-exposure to the context (extinction session). Finally, the day after extinction, rats were tested in a 5-min context re-exposure.10 is also reported as the outcome of Bayesian analyses for the estimation of posterior probability  = 14.1, p = 0.001, 10 = 37.7], there was no effect of CBD  or CBD x extinction interaction .We initially used a mild conditioning procedure that was similar in intensity to our previous work. With two mild footshocks, a moderate level of contextual freezing (35–40%) was observed at the start of the extinction session. In comparing the extinction and no-extinction conditions Figure , while tF = 7.1, p = 0.02, η2p = 0.37, BF10 = 3.2], but no observable acute effect on contextual freezing during the extinction session . Surprisingly, there was no evidence for within-session extinction . In the no-extinction control condition, planned comparisons revealed no effect of CBD at test . Therefore, CBD appears to impair the normal reduction in contextual freezing observed following extinction training in an effect that is not attributable to direct effects of CBD injection upon test performance on the next day.Planned comparison of the extinction condition revealed a significant effect of CBD to elevate contextual freezing at test , with post-hoc comparisons (p < 0.05) showing that the MK-801 group displayed higher levels of contextual freezing than both the Saline control group and the DCS-injected rats (the latter two groups not differing from each other). This difference at test was not attributable to pre-existing differences at the extinction session, with no effect of group  or group x bin  interaction. While there was a significant effect of bin , this was not driven by a decline in freezing over the course of the session, and so there was again little evidence for within-session extinction.In order to disambiguate whether the CBD-induced elevation of freezing reflects an impairment of extinction or perhaps an enhancement of reconsolidation, we administered MK-801 and DCS under the same experimental conditions. Injection of MK-801 appeared to impair the extinction of contextual freezing, whereas DCS had no observable effect Figure . ANOVA rF = 47.4, p < 0.001, η2p = 0.66, BF10 = 27.5 × 103], there was no effect of CBD  or CBD x extinction interaction . Planned comparison of the extinction condition revealed a significant effect of CBD to reduce contextual freezing at test , as well as a moderate acute impairment of contextual freezing during the extinction session . Under these conditions there was a significant reduction in contextual freezing during the course of the extinction session . In the no-extinction control condition, planned comparisons revealed no effect of CBD at test . Therefore, with stronger conditioning CBD appears to enhance the normal reduction in contextual freezing observed following extinction training in an effect that is not attributable to direct effects of CBD injection upon test performance on the next day.Given the unexpected effects of CBD observed above, in terms of our failure to replicate the reduction in contextual freezing observed previously, the lack of acute effect of CBD at the extinction session and the lack of within-session extinction, we repeated the experiment using stronger conditioning parameters involving 6 mild footshocks. In comparing the extinction and no-extinction conditions Figure , while tF = 5.31, p = 0.014, η2p = 0.34, BF10 = 4.5], with post-hoc comparisons (p < 0.05) showing that the DCS group displayed lower levels of contextual freezing than both the Saline control group and the MK-801-injected rats (the latter two groups not differing from each other). This difference at test was not attributable to pre-existing differences at the extinction session, as the effect of group  were both driven by lower freezing in the MK-801-injected rats. Analyses of the simple main effects of group at each bin revealed significant effects in the 1st and 2nd bins only , for which post-hoc pairwise comparisons confirmed that the MK-801 group was lower than the saline group, with no other differences. As with the CBD experiment, for these stronger conditioning parameters there was a significant reduction in contextual freezing over the course of the extinction session , apart from in the MK-801 group.Again, to disambiguate extinction and reconsolidation accounts of the CBD-induced reduction in freezing, we injected MK-801 and DCS in the same stronger conditioning setting. Injection of DCS appeared to potentiate the extinction of contextual freezing, whereas MK-801 had no observable effect Figure . ANOVA rHere we demonstrate that i.p. CBD and MK-801 injections 30 min prior to extinction training had a common effect of increasing subsequent contextual freezing when contextual fear conditioning was relatively weak. In contrast, CBD replicated the effect of DCS to reduce contextual freezing when conditioning was stronger. These effects of CBD were critically dependent upon the extinction training, as CBD alone had no effect upon subsequent contextual freezing. These results suggest that the long-term impact of CBD on extinction depends upon the prior conditioning experience.Under the strong conditioning procedure, CBD reduced subsequent freezing by a similar extent to DCS . In contrast, MK-801 had no observable effect. Given that DCS has previously been shown to potentiate contextual fear memory extinction , resulting in no modulation of contextual freezing. Instead, CBD replicated the effect of MK-801 to increase freezing at test. No-extinction controls confirmed that this elevation in freezing at test was not a direct effect of CBD, as might be predicted from a putative CBD-induced long-lasting attenuation of activity . The funders had no other involvement in any aspect of this work.FG is co-inventor of the patent “Fluorinated CBD compounds, compositions and uses thereof. Pub. No.: WO/2014/108899. International Application No.: PCT/IL2014/050023”; Def. US no. Reg. 62193296; 29/07/2015; INPI in 19/08/2015 (BR1120150164927). The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, we report the transfer of graphene onto eight commercial microfiltration substrates having different pore sizes and surface characteristics. Monolayer graphene grown on copper by the chemical vapor deposition (CVD) process was transferred by the pressing method over the target substrates, followed by wet etching of copper to obtain monolayer graphene/polymer membranes. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and contact angle (CA) measurements were carried out to explore the graphene layer transferability. Three factors, namely, the substrate roughness, its pore size, and its surface wetting (degree of hydrophobicity) are found to affect the conformality and coverage of the transferred graphene monolayer on the substrate surface. A good quality graphene transfer is achieved on the substrate with the following characteristics; being hydrophobic (CA > 90°), having small pore size, and low surface roughness, with a CA to RMS (root mean square) ratio higher than 2.7°/nm. It is very strong (100 times stronger than steel) , highly  copper) , has a hr) [2/g) , is highr) [2/g) ,6, is hir) [2/g) ,8, and fr) [2/g) ,10,11.With the aforementioned unusual properties, graphene opens doors for many applications across disciplines. It is used in electronic applications as transistors , chemicaVarious fabrication routes exist for graphene production, including the mechanical ,28 and cAt present, no single fabrication route that produces graphene sheets is suitable for all potential applications, as every route has its advantages and disadvantages ,37. GrapCurrently, graphene transfers onto desired substrates using various methods are implemented in two ways: the wet transfer method and the dry transfer method. The most straightforward wet transfer method is to etch the metal away chemically to obtain free floating graphene composites that can be scooped onto chosen substrates. Other well-known wet etching methods include the standard transfer method , the dirIn all wet transfer methods, the Cu substrate is removed by a wet etching process using a copper etchant such as ferric chloride , iron niBunch et al. , revieweIn the present work, we examine the effect of substrate surface characteristics in terms of pore size, wettability (degree of hydrophobicity), and surface roughness on the graphene transferability experimentally. We selected eight polymeric substrates with different surface characteristics and transferred monolayer CVD grown graphene on copper foils onto these polymeric substrates. Copper was removed by wet etching to obtain monolayer graphene/polymer composite membranes. Two conditions related to the substrate surface were found to affect the quality of transferred graphene. Firstly, the substrate should have an adequate hydrophobicity (contact angle (CA) > 90°); if the first condition is achieved, then the ratio of the surface contact angle to the root mean square (RMS) value should be roughly higher than 2.5. Otherwise, poor graphene quality will be obtained. The quality of graphene was checked by Field Emission Scanning Electron Microscope (FESEM) and Atomic Force Microscopy (AFM) characterization, and an ionic transport study of potassium chloride (KCl) through the graphene/substrate composite membranes.We transferred monolayer graphene onto various polymeric substrates by modifying the direct transfer method developed by Regan et al. . It is aThe quality of transferred graphene was checked by studying the ionic transport through the graphene/substrate composite. To do so, a special Side-bi-Side glass diffusion cell procured from Permegear Inc., Hellertown, PA, USA was used . The celBoth cell chambers are throughly cleaned by de-ionized water and air dried before the composite graphene membrane is placed between them. The active side of the composite membrane (graphene side) faces the left chamber. The cell is then tightened using a rubber screw. Both sides of the membrane are then washed with ethanol to remove any water bubbles close to the membrane surfaces. To remove any entrapped ethanol from the previous stage, the left cell side was washed with 0.5 M KCl solution and the right side was washed three times with de-gassed de-ionized water.The ionic transport study was performed with 7 mL of 0.5 M KCl solution. KCl solution was introduced into the left chamber of the cell and 7 mL de-ionized water was introduced into the right chamber. Both solutions were magnetically stirred during the diffusion process to minimize concentration polarization effects. Potassium and chloride ions diffused through the graphene membrane towards the de-ionized water side. The diffusion rate of the ions was measured by monitoring the change in conductivity over time, using an eDAQ conductivity isoPod electrode  dipped in the de-ionized water side of the diffusion cell, as shown in FESEM, AFM, and water wettability characterizations for the as received polymer membranes and the graphene/polymer membranes were carried out to understand the role of the substrate surface characteristics on the graphene transferability as well as its coverage and quality.2. However, for PVDF 6 substrate, an area of 4 × 4 µm2 was used as AFM tip instability problems were encountered due to a rather rough surface for this particular substrate. Three sections were taken to explore the surface profile and to calculate the average root mean square (RMS) values. 3D surface profiles were also captured to check and qualitatively validate surface roughness values.The AFM results show that the PVDF 6 substrate with 100 nm pore size was found to have the roughest surface of RMS = 112 nm and appreciably high surface wettability of CA = 109° as shown in It is not always necessarily true that smaller pore size substrates should have smoother surfaces. PVDF 4 shown in Based on the contact angle measurements, four out of the eight substrates, namely, PES, PVDF 1, PVDF 2, and PVDF 3 are considered hydrophilic; (CA < 90°), and the remaining are considered hydrophobic.We transferred monolayer graphene onto polymeric substrates using the procedure (pressing method) shown in Graphene detached from four substrates  during copper etching as mentioned earlier (failed transfer). The contact angle of these substrates is lower than 90° and as such they can be considered hydrophilic. To prevent graphene layer detachment, a polymeric substrate with an adequate degree of hydrophobicity is required to prevent etchant penetration during the etching process that usually takes around 2 h.The other four substrates  have the required degree of hydrophobicity that prevents the detachment process; PVDF 2 has an average CA = 90° and PVDF 4 has a CA = 116°. Two of the four substrates (PP and PVDF 5) have smoother surfaces compared to the PVDF 4 and PVDF 6. The PP has a surface roughness with a root mean square (RMS) value = 42.4 nm; PVDF 5 has an RMS value = 23.8 nm. The other two substrates have a rougher surface, with 94.9 nm and 112.1 nm RMS values for PVDF 4 and PVDF 6, respectively .2 of monolayer graphene can be clearly seen by the naked eye at the upper right corner (inset). The FESEM images , the KCl ions are transported from the left side toward the right side through the graphene/polymer composite membrane. The rate of ion transport is calculated by monitoring the change of de-ionized water conductivity over time. Based on the fact that a defect free graphene is impermeable even to very small ionic species such as helium ,56, the So far, two conditions related to substrate surface characteristics should be controlled in order to have a good quality (low defect density) graphene transfer. First, the substrate surface should have a reasonable hydrophobicity (contact angle > 90°) and second, the contact angle (CA) to surface roughness  should be higher than 2.7 (CA/RMS > 2.7°/nm).A hydrophobic surface along with a high CA/RMS ratio means the substrate has the required hydrophobicity to prevent penetration of the etchant between the Cu/graphene and substrate, and the low roughness helps provide better contact between the transferred graphene and substrate, and minimize the unsupported graphene domains that could be detached during the etching process .Graphene transfer to a working substrate is the key to a wide range of graphene applications. This paper explores the effect of substrate surface characteristics, mainly the surface roughness and wettability (degree of hydrophobicity) on graphene transferability. To transfer graphene with high quality, the substrate surface should be smooth to allow the graphene to conform and adhere with minimum tears and cracks, and should have high hydrophobicity to prevent etchant penetration between the graphene and substrate during copper etching, which would otherwise cause graphene detachment. CVD monolayer graphene was transferred to eight different polymeric substrates having different surface characteristics by a simple pressing method, followed by wet etching of copper using an APS etchant. Graphene failed to transfer over four substrates  because they were hydrophilic, which facilitated etchant penetration between the Cu/graphene and the substrate and caused graphene detachment. Graphene successfully transferred to the other four substrates that were hydrophobic . PP and PVDF 5 exhibited better graphene transferability in terms of quality, due to the lower surface roughness as compared to PVDF 4 and PVDF 6. The quality of the graphene was checked by FESEM characterization and the simple diffusion of potassium chloride ions (KCl) through the transferred graphene/substrate membrane. To obtain high-quality graphene, the substrate should have an adequate hydrophobicity (contact angle (CA) > 90°); if this is attained, then the ratio of the contact angle value to the root mean square (RMS) value should be higher than 2.7 (CA/RMS > 2.7°/nm).


Second, in cell lines that express high levels of PD-L1 , the ratio of p-YAP/YAP was lower and GTIIC reporter activity of the Hippo pathway was higher than those in three cell lines expressing low levels of PD-L1  (P < 0.05). Third, in the same three cell lines, inhibition of YAP by two small interfering RNAs (siRNAs) decreased the mRNA and protein level of PD-L1 (P < 0.05). Fourth, forced overexpression of the YAP gene rescued the PD-L1 mRNA and protein level after siRNA knockdown targeting 3′UTR of the endogenous YAP gene. Finally, chromatin immunoprecipitation (ChIP) assays using a YAP-specific monoclonal antibody resulted in the precipitation of PD-L1 enhancer region encompassing two putative TEAD binding sites. Our results indicate that YAP regulates the transcription of PD-L1 in NSCLC.Programmed death-ligand 1 (PD-L1) is a membrane protein on tumor cells that binds to the PD-1 receptor expressed on immune cells, leading to the immune escape of tumor cells. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signaling pathway, which plays important roles in cancer development. Here we show that YAP regulates PD-L1 expression in human non-small cell lung cancer (NSCLC) cells. First, we investigated YAP and PD-L1 expression at the protein level in 142 NSCLC samples and 15 normal lung samples. In tumor tissue, immunohistochemistry showed positive staining for YAP and PD-L1, which correlated significantly ( Lung cancer remains the leading cause of cancer-related mortality worldwide , 2, and PD-L1 , a type I transmembrane surface glycoprotein encoded by the CD274 gene, promotes T-cell tolerance and escapes host immunity by inhibiting CD8+ T-cell survival, effector function, and inducing Fas-mediated T-cell apoptosis . PD-L1 iet al. [Overexpression of the transcription coactivator Yes-associated protein (YAP) has been found in many cancers due to abnormal amplification, loss of Hippo signaling by mutation, and/or downregulation of the core Hippo component . YAP oveet al.  indicateet al. . AlthougIn this study, we hypothesized that YAP is involved in the regulation of PD-L1 expression in NSCLC. To test this, we evaluated YAP and PD-L1 expression in human NSCLC tissues and then investigated whether PD-L1 is a downstream target of YAP in NSCLC cell lines.s = 14.947, P < 0.001). In normal lung tissues, no cases were positive for YAP or PD-L1 (Table P > 0.05) .YAP is an important mediator protein in cancer development. Programmed death-ligand 1 , which is expressed on many cancer cells, is one of the most important inhibitory molecules that promote tumor immune escape. To investigate the relationship of YAP and PD-L1 in NSCLC tissues, we used immunohistochemistry (IHC) to examine their protein level in 142 samples of primary human NSCLC and 15 normal lung samples Figure . Forty-t > 0.05) . To furtP < 0.05; Figure P < 0.05; Figure P < 0.001), but YAP mRNA expression was lower than that in SKLU-1 and H1299 cell lines (P < 0.001), higher than that in A549, H2170 and H2030 cell lines (P < 0.01), and the same as that in H1975 and PC9 cell lines   was used to silence the YAP gene in H460, SKLU-1 and H1299 cell lines. The mRNA and protein levels of PD-L1 were detected, respectively, by qRT-PCR and Western blot. The results confirmed that the YAP gene was significantly inhibited by transfection of siRNA-YAP Figure . qRT-PCR) Figure . Western) Figure . After Y) Figure . TogetheP < 0.01)  than in other cell lines. Moreover, in H460, SKLU-1 and H1299 cells, inhibition of the endogenous YAP by siRNAs downregulated PD-L1 mRNA and protein levels, and this could be rescued by forced overexpression of YAP. Third, our ChIP results demonstrated that precipitation of the PD-L1 enhancer region encompasses the putative TEAD binding site in H460, SKLU-1 and H1299 cell lines.P > 0.05). In tumor development, YAP is thought to be associated with a variety of factors. A significant correlation between YAP and Jag-1 staining was observed in pancreatic ductal adenocarcinoma  [P < 0.001). Expression of the two proteins was mildly, but still significantly correlated .In our study, YAP and PD-L1 were expressed to different degrees in NSCLC tissues. YAP, a candidate oncogene in a variety of cancers, is significantly elevated in hepatocellular cancer, prostate cancer, and breast carcinoma , 23. The< 0.001) . Howeveret al. [et al’s western blot results. In their study, PD-L1 expression in H1975 cells was also stronger than in PC9 cells. We also found that high expression of PD-L1 is positively correlated with an increase of intranuclear YAP. YAP activation plays an important role in transcriptional regulation. Phosphorylation of YAP by LATS1 leads to cytoplasmic retention of YAP, which inhibits its function as a nuclear transcription coactivator [We investigated eight NSCLC cell lines and found that three  had higher PD-L1 expression in mRNA and protein level compared to others. Chen ctivator –32. In cYAP translocation into the nucleus, as a transcriptional coactivator, plays an oncogenic function mainly by interacting with the TEAD family of transcription factors , 34. Accet al. [Expression of PD-L1 as an adaptive response to endogenous antitumor immunity can occur because PD-L1 is induced on most tumor cells in response to inflammatory cytokines, predominantly IFN-γ, which has been confirmed in melanoma, ovarian cancer, and part of lung cancer . IFN-γ iet al.  showed tet al. . Recentlet al. . FurtherIn conclusion, our study indicates that YAP and PD-L1 expression are significantly correlated in NSCLC tissues. The reason for this may be that YAP regulates the expression of PD-L1 in NSCLC at the transcriptional level. Future studies should focus on whether regulation of YAP and PD-L1 is active in a variety of tumor types and whether this should affect the choice of clinical treatment and prognosis for NSCLC in the future.2.Human NSCLC cell lines H460, H2170, SKLU-1, H1975, H1299, A549, H2030, and PC-9 were obtained from American Type Culture Collections . Cell lines were maintained in RPMI-1640 except for SKLU-1, which was maintained in EMEM. All media were supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 mg/ml). LP-9 was maintained in M199 supplemented with 15%(v/v) heat-inactivated FBS, 10 ng/ml EGF, 0.4 ug/ml hydrocortisone and penicillin (100 IU/ml). All cells were cultured at 37°C in a humid incubator with 5% COTotal RNA was extracted from cells using the RNeasy Mini kit . The cDNA was transcribed from 500 ng of total RNA using iScript-cDNA Synthesis Kits , according to the manufacturer’s protocol. The cDNA was used as the template for real-time PCR detection using TaqMan Technology on an Applied Biosystems 7000 sequence detection system . Expression of PD-L1, YAP genes and endogenous control gene b-glucuronidase (GUSB) was detected by using the primer and probe sequences commercially available  and analyzed using Relative Quantification Software (Applied Biosystems). The TaqMan Assay IDs of primer sequences for qRT-PCR were GUSB , PD-L1  and YAP1 . Waltham, MA USA 02451.The SMAR-Tpool siRNA targeting YAP (YAP siRNA-1) was purchased from Thermo Scientific Dharmacon . Non-targeting siRNA was used as control (Thermo Scientific Dharmacon). Another YAP siRNA targeting the 3′UTR end of the YAP gene (YAP siRNA-2) was purchased from Life Technologies . The YAP plasmid DNA used to overexpress the YAP gene in the cells was purchased from Addgene . Cells were plated in 6-well plates (for western blot) and 24-well plates (for PCR and reporter assay) for 24 hours before treatment. Cells were transfected with 4μg of YAP plasmid DNA using Lipofectamine 2000  transfection reagent, and 100 nmol/L of siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. After transfection for 48 hours, cells were harvested for further analysis.The 8 × GTIIC-luciferase plasmid (Addgene) and Renilla luciferase pRL-TK plasmid  were co-transfected into cell lines. The si-RNA transfection reagent was Lipofectamine RNAiMAX (Invitrogen). After 48 hours, cells were harvested and transferred into a 96-well plate for analysis by using the Dual-Luciferase Reporter Assay Kit (Promega). Luminescent signaling was detected on a GloMax-96 Microplate Luminometer (Promega) according to the manufacturer’s instructions.The following primary antibodies were used for immunoblot analysis: YAP, phospho-YAP (Ser127), Src, Lamine-b and PD-L1 from Cell Signaling, Inc. ; phospho-YAP (Tyr357) from Abcam  and Sigma-Aldrich ; TAZ and α-tubulin from Santa Cruz Biotechnology . Total protein was extracted from cell lines using M-PER Mammalian Protein Extraction Reagent , and nuclear/cytoplasm proteins extracted using a nuclear/cytoplasm extraction kit (Thermo Fisher Scientific Inc.) were supplied with Complete Protease Inhibitor Cocktails , according to the manufacturers’ protocols. The protein concentrations were measured with the Pierce BCA Protein Assay Kit . A total of 15 μg of proteins was run on 4∼20% gradient SDS–polyacrylamide gels (Bio-Rad) and transferred to Immobilon-P nitrocellulose membranes . The membranes were blocked in 5% non-fat milk and then probed with the primary antibodies overnight at 4°C. The membranes were incubated with appropriate secondary antibodies, and detected by using an ECL blotting analysis system .Fresh lung tumor tissues were obtained from patients who were undergoing surgical resection of a primary tumor. All human tissue samples were obtained and analyzed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714–22 942–01). The tissue microarray sections were immunostained as previously described . The folCells were seeded on a Chamber SlideTM (Thermo Fisher Scientific) at 50% confluence and fixed with 100% methanol following their respective treatments. After permeabilization using Triton-X100, slides were subsequently blocked for 1 h at room temperature with Dulbecco’s phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) and incubated with primary antibody overnight at 4°C. Antibodies were diluted in PBS containing 5% BSA. Primary antibodies of YAP and phospho-YAP (Ser127) were purchased from Cell Signaling, Inc. . After being washed, the slides were incubated with the corresponding secondary antibodies in the dark for 1 h at room temperature, washed again, and mounted using ProLong Antifade with DAPI to visualize the nuclei. The slides were analyzed using a fluorescent confocal microscope equipped with an ultraviolet laser .Fragmented chromatin from SKLU-1 and H1299 cells was incubated with anti-IgG (negative control), anti-YAP, and anti-POL-II (positive control). Fragmented chromatin from SKLU-1, H1299, H1975 and A549 cells was incubated with anti-YAP. Recruited DNA was subjected to PCR using the primers for distal enhancer regions of PD-L1, and PCR products were electrophoresed in agarose gel. The ChIP assay was conducted using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore Corporation). Monoclonal antibodies for YAP  and control rabbit antibody for IgG  were used for ChIP. Two pairs’ primer used for RT-PCR to amplify the PD-L1 gene, one pair primers were 5′-TCGGTCTGTGAAGGACTGC-3′ and 5′-ACCGTTGAGGAATGGATGAA-3′ resulting in a product size of 203 bp, and the other were 5′-CCACCACCATTATCTAATTCCA-3′ and 5′-AAGGAGCCAGACACAAAAGG-3′ resulting in a product size of 210 bp.t-test. The relationship between YAP and PD-L1 was analyzed with Spearman product correlation. For all analyses, statistical significance was defined as P < 0.05 , based on two-tailed tests.Data are expressed as mean ± standard deviation (SD) from independent experiments. All statistical analyses were performed using the SPSS 23.0 for Windows software system . One-way ANOVA followed by Scheffe multiple comparisons were used to compare the differences among multiple groups. Statistical significance of differences between groups was determined by Student’s


The PD-1 receptor and its ligands PD-L1 and PD-L2 are known to be significantly involved in T-cell regulation. Recent studies suggest that PD-L1 expression in malignant tumors contributes to an immunosuppressive microenvironment and disruption of antitumoral immune response. Drugs targeting this pathway are already tested in clinical trials against several tumor entities with promising results. However, until now comprehensive data with regard to PD-L1 and PD-L2 expression in head and neck squamous cell carcinoma (HNSCC) is still lacking.We assessed PD-L1 and PD-L2 expression via immunohistochemistry in two independent cohorts of 293 HNSCC patients.A significant subset of HNSCC showed high expression levels of PD-L1. Most remarkable, we detected a strong correlation between PD-L1 expression and overall survival time in both HNSCC cohorts. Further, in multivariate cox proportional hazard models, PD-L1 dominates as the strongest prognostic factor of patient's outcome in HNSCC, leaving even tumor stage and distant metastasis behind. Moreover, strong PD-L1 expression was associated with the presence of distant metastases in a subset of cases.In summary, while the significance of PD-L2 in HNSCC seems to minor, we show that PD-L1 expression is common in HNSCC and, more importantly, a both robust and strong prognostic biomarker. In this respect, our results provide hints on further application of therapies targeting the PD-1/PD-L1 pathway in HNSCC. Investigation of response and outcome of patients receiving anti-PD-1/PD-L1 containing therapies in correlation with PD-L1 expression analysis should be an important task for the future.In spite of improved treatment options and increasing knowledge of molecular alterations in HNSCC, the survival rate has not been dramatically changed in the past decades. Pies are missing in HNSCC. One promising candidate in cancer immune therapy is PD-L1.Drugs targeting PD-L1 or its receptor PD-1 are subject of several clinical studies in different cancer entities. However, comprehensive data about PD-L1 expression in HNSCC and therefore a rational basis for anti PD-L1/PD-1 therapy in HNSCC is lacking. Here, we provide wide-ranging data about PD-L1 expression in HNSCC of all major localizations. We observed a strong correlation between expression of PD-L1 and reduced overall survival time. Furthermore, high PD-L1 expression was identified as a strong prognostic factor of patient's outcome when verified together with recognized prognostic factors. Additionally, most clinical trials investigating anti-PD-1/PD-L1 drugs use proprietary antibodies. Recently, we demonstrated that the utilization of the novel monoclonal rabbit antibody against PD-L1 (clone EPR1161) on FFPE tissue is specific and independent of the used detection system . NotablyIn summary, we are the first to demonstrate the prognostic value of PD-L1 expression in HNSCC of all major localizations. On the basis of our results, it seems likely, that HNSCC with a high PD-L1 expression and putative activation of the PD-1/PD-L1 pathway represent a highly aggressive cancer phenotype, independent of tumor origin, tumor stage or grade. Perhaps, treatment-related acute and late toxicity of conventional radiochemotherapy could be reduced by complementary therapies targeting the PD-1/PD-L1 pathway in the future , 18, 32.This study includes two independent cohorts of paraffin-embedded primary HNSCC specimens, which were obtained from the archive of the Institute of Pathology, University of Bonn Medical Center . Two pathologists histologically confirmed the tumor diagnosis.The first HNSCC cohort (training set) consists of 98 patients, in detail 16 female (16%) and 82 male (84%) patients. The median age was 64 years, ranging from 38 to 88 years. In the second cohort (testing set), 53 female (27%) and 142 male (73%) patients were included, the age distribution ranged from 27 to 82 years. The median age was 62 years. Both cohorts included tumors derived from the oral cavity, oropharynx, hypopharynx and larynx. Patients included in the study had a localized or locally advanced disease. All patients were treated by surgery with optional adjuvant radio-chemotherapy or definitive radio-chemotherapy with curative intent. All investigations were conducted in an anonymous manner, as approved by the local ethics committee at the University Hospital of Bonn .TMAs were constructed as previously described . BrieflyParaffin-embedded tissue samples were cut in sections of 3 μm and mounted on Super Frost Plus slides . Slides were routinely processed, deparaffinized in xylene and rehydrated in a degraded alcohol sequence. Immunohistochemistry was performed on Ventana BenchMark Ultra automated staining system  and visualized with the Ventana amplifier detection kit using PD-L1, clone EPR1161 (2) antibody  and PD-L2, clone 176611 antibody  Slides were counterstained with hematoxylin. Following dehydration in a graded alcohol sequence and steadied in pertex® . Validation of the specificity of this PD-L1 antibody was performed previously .Immunohistochemical stainings were examined independently by two pathologists , who were blinded to patients' clinical outcome. Membranous and cytoplasmic staining of tumor cells was considered. The first HNSCC cohort served as a training set for identifying suitable PD-L1 and PD-L2 expression thresholds for subsequent statistical analyses. As a result, PD-L1 and PD-L2 expression was scored semiquantitatively as negative (0), low (1) or strong (2) in both HNSCC cohorts. In detail, cases with no expression were scored “negative”, specimen with only minor intensity were scored “low”, and cases with a strong expression were scored “high”. To our experience, cytoplasmic PD-L1 expression was only detected in combination with a membranous staining in the same tumor cell. Vice versa, there were tumor cells which only displayed a membranous staining, and no cytoplasmic PD-L1 expression. We did not observe tumor cells with solitary cytoplasmic PD-L1 expression. We previously validated the specificity of the PD-L1 antibody in a preceding study .Statistical analyses were carried out using the SPSS 22 software package . PD-L1 protein expression was correlated with clinical-pathological data available using Chi-Square test or Pearson correlation. Overall survival time was defined as the time between first diagnosis and death of the patient or last clinical follow-up if the patient was still alive, respectively. Survival time was estimated by Kaplan-Meier analyses and compared among patient subsets using log-rank tests. Protein expression was also examined within univariate and multivariate Cox proportional hazards regression models. All statistical tests were two-sided. P-values <0.05 were considered to be statistically significant.


TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages. TDP‐43 is a ubiquitously expressed, predominantly nuclear, RNA binding protein, which is involved in multiple steps of RNA processing and maturation, including transcription and splicing  of ALS cases; however, cytoplasmic TDP‐43 inclusions, accompanied by nuclear depletion of the protein, are the key pathological hallmark of > 98% of ALS indicating the profound importance of TDP‐43 in pathogenesis  by which TDP‐43 mutations lead to disease remains unknown, and both nuclear loss of function (LOF) and cytoplasmic gain of function (GOF) have been proposed to play a role  or mutant TDP‐43 cause multiple RNA changes, making it difficult to identify the pathogenic RNA profile that occurs physiologically in disease  of TDP‐43, which induces a splicing GOF. The analysis of this GOF uncovered entirely novel splicing events in genes not known to be controlled by TDP‐43, such that a set of constitutive exons—here named “skiptic exons” (SE)—are skipped due to TDP‐43 GOF.Here, we have addressed these issues by working with animals from an allelic series carrying point mutations within mouse endogenous skipping of constitutive exons induced by GOF is in contrast to the previously identified TDP‐43 LOF‐induced cryptic exons, which are normally repressed but in LOF are aberrantly included in mRNAs  mouse mutagenesis programmes . nockdown , showingnockdown B. Surpri GOF Fig B.Sortilin 1. Exon 18 inclusion was increased in RRM2mut, as previously described in TDP‐43 knockdown  comparing them to scramble sequence shRNA  and remarkably had only one significant splicing change and eight differentially expressed genes (FDR < 0.05)—indicating that RRM2mut LOF and LCDmut GOF complement each other and that the combination of mutations results in viable animals  and to define them we used the following per cent spliced in (PSI) criteria—exons that were included in WT (PSI > 0.95) and skipped in mutants (∆PSI decrease of > 0.05).We further analysed the skewed preference in LCDmut RNA‐seq data and validated 7 with RT–PCR. Mean and median PSI of these events in wild‐type littermates were 0.98 and 0.99, mean and median ∆PSI in LCDmut were −0.12 and −0.09, and LCDmut exon exclusion mean and median fold‐changes were 17.6 and 8.6, respectively , in which TDP‐43 LOF induces the inclusion of otherwise constitutively repressed exons  and LOF affects a separate set of genes where it induces the inclusion of CEs Fig .RRM2mut  in 15/48 transcripts, resulting in likely unstable transcripts. Therefore, we compared expression of transcripts containing SEs to transcripts carrying other differentially regulated cassette exons, and all other transcripts expressed at similar levels in our RNA‐seq dataset; we found the transcripts that included SEs were significantly more downregulated  from 18% in SEs showing no expression change to 54% in significantly downregulated SEs  counts performed on the sciatic MN pool at L4–L5 showed a 28% reduction in homozygous LCDmut when compared to littermate controls  model via CRISPR/Cas9 genome editing carrying a Q331K mutation  and M323K (LCDmut) for further analysis, which were initially on a hybrid DBA/2JxC57BL/6J background. Mice were rederived via in vitro fertilisation (IVF) using frozen F1 sperm at RIKEN and maintained on C57BL/6J and DBA/2J backgrounds at MRC Harwell.The RIKEN and MRC Harwell ENU‐DNA archives were screened for coding mutations within All animals were housed and maintained in the Mary Lyon Centre, MRC Harwell, under specific opportunistic pathogen‐free (SOPF) conditions, in individually ventilated cages adhering to environmental conditions as outlined in the Home Office Code of Practice. All animal studies were licensed by the Home Office under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 (SI 4 2012/3039), UK, and additionally approved by the Institutional Ethical Review Committee. Mice were euthanised by Home Office Schedule 1 methods.Initial genotyping for the Q331K mutation was performed as follows: genomic DNA from F0 and F1 animals was extracted from ear clip biopsies using DNA Extract All Reagents Kit (Applied Biosystems). The targeted region was PCR‐amplified using high‐fidelity Expand Long Range dNTPack (Roche) and genotyping primers Geno_Tardbp_F1/R1. PCR products were further purified using gel extraction kit (Qiagen) and analysed by Sanger sequencing. PCR product amplified from DNA obtained from F0s that showed the correct point mutation (C>A inducing Q331K) was sub‐cloned using Zero‐Blunt PCR cloning Kit (Invitrogen), and 12 clones were analysed by Sanger sequencing.et al, A grip strength meter (BioSeb) was used to measure muscle strength. Total limb (fore and hind) were measured twice with an average value recorded, as previously (Ricketts Tardbp and human TARDBP (TGGATGAGACAGATGCTTC) in parallel with scramble control. In order to improve the percentage of MEFs expressing shRNA, 3 days after transfection, MEFs underwent 3‐day treatment with puromycin at 4 μg/ml and 3 days at 2 μg/ml before being harvested in RIPA buffer for protein analysis and Trizol for RNA extraction. Tardbp levels were validated using Tardbp FAM TaqMan probes along with Gapdh and Actb VIC probes in separate reactions.Mouse embryonic fibroblasts were derived following established protocols from E14.5 embryos derived from intercrosses to obtain all genotypes within one generation, allowing to use littermates as controls. Primary cells were not tested for mycoplasma. In order to obtain TDP‐43 knockdown in MEFs, we used shRNA from the GIPZ Lentiviral shRNA library (GE Dharmacon) with the following sequence shared between mouse et al, Tardbp genotypes. Briefly, an Amaxa system (Lonza) was used following manufacturer's instructions. After 24 h, MEFs were harvested and cell pellets snap‐frozen. For this and any other validation of any splicing event, RNA was extracted following standard procedures via Trizol (Life Technologies) or columns (Qiagen), and cDNA produced via RT–PCR following manufacturer's instructions . CFTR exon inclusion was assessed via semiquantitative RT–PCR using primers specific against the minigene construct.Electroporation was used to transfect the CFTR minigene construct  or UG6 repeats were used for different assays. The EMSA gel (5% acrylamide) was loaded with the recombinant protein in binding buffer together with γ32P labelled RNA (UG6 repeats or Tardbp CLIP sequence). After the run was completed, the gel was dried (583 Gel Dryer Bio‐Rad) and visualised using X‐OMAT film or autoradiographic XAR film (F5763 Sigma/Kodak).Mouse TDP‐43 WT and the different mutations were expressed in RRM2mut cryptic n = 4, LCDmut skiptic n = 4, Q331K splicing/skiptic n = 3). Products were electrophoresed on agarose gels with ethidium bromide (Sigma) or using D1000 ScreenTape and reagents (Agilent) on the 2200 Tapestation system (Agilent). Results were analysed using PSI, calculated dividing the intensity of the exon inclusion band by the sum of both exon inclusion and exon exclusion bands. When analysing SEs in human fibroblasts, as a combination of three primers was used, we did not calculate PSI, but calculated the ratio between SE band and the exon inclusion band and expressed results after normalizing the mean of healthy controls to 100%. Primer sequences are available upon request.For validation of splicing events from different sources, RNA was extracted from MEFs, embryonic E18.5 head or adult spinal cord. RNA quality was determined by RNA Integrity Number (RIN) measured using RNA ScreenTape and reagents (Agilent) on the 2200 Tapestation System (Agilent). RT–PCR protocol was conducted following manufacturer's instructions . PCR for splicing events was conducted using 2X PCR Master Mix (Thermo) with specific primers spanning the differentially expressed exon .RNA‐seq was performed using polyA libraries as previously described (Wang All sequencing data are deposited in the NCBI Sequence Read Archive (reference: SRP133158).et al (RRM2mut) or adult brain (LCDmut) were homogenised in phosphate‐buffered saline (PBS) and UV‐crosslinked 4× at 100 mJ/cm2 at a wavelength of 254 nm . Protein concentrations were normalised using the DC Protein Assay (Bio‐Rad), and the lysate was then sonicated for 30 s ×10 times. Lysates were then treated with 0.1 U/μl RNase I (Thermo) and Turbo DNase (Thermo). Anti‐TDP‐43 antibody (Sigma) was conjugated to Dynabeads Protein A (Thermo) and used to immunoprecipitate TDP‐43 from the tissue lysate. The 3′ ends of attached transcripts were then dephosphorylated using T4 PNK (NEB) and the pre‐adenylated L3‐App adaptor ligated using RNA ligase (NEB). The 5′ end of transcripts were labelled with radioisotope ATP[γ−32P] using T4 PNK. The labelled complexes were then resolved on NuPAGE 4–12% Bis‐Tris gels (Invitrogen), transferred to nitrocellulose membrane and exposed to Fuji film. The protein–RNA complexes corresponding to TDP‐43 size of 43 kDa and above were isolated, and the protein was digested with proteinase K (Sigma). RNA fragments were reverse‐transcribed using SuperScript III (Invitrogen) and size‐resolved on Novex 6% TBE‐urea gels (Invitrogen). Bands of size 75–95 nt and 95–200 nt were excised, further purified, circularised using Circligase (Epicentre) and digested with BamHI. cDNA fragments were amplified by PCR and further purified with Ampure XP beads (Beckman Coulter). The iCLIP libraries were quantified using the D1000 ScreenTape and reagents (Agilent) on the 2200 Tapestation System (Agilent) and sequenced using the standard Illumina protocol for 50‐bp single‐end sequencing , TDP‐43  and ubiquitin  was conducted using a Ventana immunohistochemical staining machine (Ventana). Following staining with the appropriate secondary antibodies, the sections were developed with 3,3‐diaminobenzidine. All experiments were carried out using a positive [human frontotemporal dementia (FTD) cortex] and negative control.For analysis of spinal cord pathology, 1‐ and 2‐year‐old WT, Spinal cords from 2‐year‐old LCDmut HOM mice were homogenised in RIPA buffer with protease inhibitors. The homogenate was resolved using Western blot using NuPAGE 4–12% Bis‐Tris gels (Invitrogen) using anti‐TDP‐43 (Proteintech). Quantification of the results was conducted on the Odyssey infrared imaging system (Li‐Cor).et al,Briefly, turbidity measurements were obtained on WT and mutant TDP‐43 C‐terminal fragments (267–414) thawed from frozen stock, desalted into 20 mM MES (pH 6.1) and diluted to 20 μM in a 100 μl final volume in 20 mM MES supplemented with 0–500 mM NaCl , transcardially perfused with 4% PFA and the lumbar region of the spinal cord removed, post‐fixed in 4% PFA and cryopreserved in 30% sucrose. Serial 20‐μm transverse sections were cut on a cryostat, stained with gallocyanin, a Nissl stain, and the number of positively stained motor neurons in the sciatic motor pool of every third section between the L4 and L5 levels of the spinal cord was counted, as previously described (Joyce LCDmut littermates (N = 5) were examined at 24 months of age, as described previously :CCCGACTGGTTCTGCTGGCTGGCTAACATGCCCATCATACCCCAACTGCTCTTCAACGCTGCCTGAGCCGCAGCCATCATCGCTGGGTTAATGCTAAAAGCACCAAAGTTCATCCCTCCACCCATATTACCACCCTGGTTATTTCCCAAGCCAGCTCCACCCCCTCTACTGTTACCAAACCCACCCTGATTCCCAAAGCC.l‐glutamine until fibroblasts grew out from the explants. When confluent, fibroblasts were detached, further expanded and underwent cryopreservation. They were maintained in DMEM with 10% FBS and 1% penicillin/streptomycin under standard culture conditions. Clinical information and genetic changes are summarised in Fibroblasts were obtained from a 4‐mm skin punch biopsy taken following informed consent under local anaesthetic. Biopsies were dissected into ~1‐mm pieces and cultured in DMEM, 10% FBS, 1% Transcriptomics analysis methods are described in detail in Conceptualisation: PF, EMCF, AA‐A. Methodology: PF, PSi, JH, KL, TR, HO, AEC, YG, ML, LT, FB, LG, EB, VP, EMCF, AA‐A. Formal analysis: PF, PSi, JH, KL, TR, HO, JMB‐A, BK, YY, ML, AEC, WE, GC, EPa, PSt, EW, DEH, FB, LG, EB, VP, EMCF, AA‐A. Investigation: PF, PSi, TR, HO, JMB‐A, BK, AU, YY, NB, CB, TC, AEC, AMM, AM‐G, MS, JM, SC, WE, GC, MG, RF, YG, EPa, EPe, PSt, MH, ACa, ACh, AMI, EW, EB, AA‐A. Writing original draft: PF, AA‐A. Writing, review and editing: PF, PSi, JH, KL, TR, HO, EB, AMI, NLF, FB, EB, VP, EMCF, AA‐A. Supervision: PF, EB, VP, EMCF, AA‐A.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Source Data for Expanded ViewClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 7Click here for additional data file.


PD-L1 is thought to play an important role in the antitumor immune response. In this study, we investigated the expression of PD-L1 within breast tumor subsets to better define its prognostic significance.Immunohistochemistry was performed to determine PD-L1 tumor cell expression and to enumerate CD8, CD4 and CD68 tumor-infiltrating leucocytes (TIL) in a cohort of 443 breast cancers categorized by molecular subtype.p = 0.018). Combined PD-L1/epithelial CD8 positive status was also strongly associated with better RFS and OS  in the Basal-like subgroup.Across the entire cohort, PD-L1 tumor cell expression was observed in 73/443 (16.5%) cases and associated with known indicators of poor prognosis, including low patient age, high tumor grade, ER/PR negative status, but not with outcome. However, in the Triple Negative breast cancer subset PD-L1 was associated with better recurrence free survival (RFS) especially within the Basal-like subset (Hazard ratio = 0.39, 95% CI = 0.22 - 0.86, PD-L1 expression is associated with better patient survival in Basal-like breast cancer. PD-L1 B7-H1, CD274) is the ligand for Programmed death-1  and is expressed on the surface of cancer cells in addition to its expression on infiltrating immune cells. PD1 is an immune-suppressive receptor that is expressed on activated T cells, B cells, monocytes and dendritic cells 74 is the, 4.Recent research has shown that blockade of the interaction between PD-L1 and PD-1 can enhance T cell function and facilitate antitumor activity, and various monoclonal antibodies against PD-1 and PD-L1 are in clinical trials for a variety of solid tumor types, including breast cancer, with encouraging activity in many cancers . PD-L1 eThese conflicting results warrant further exploration. When reviewing the literature, we observed that few previous studies had been conducted on the Basal-like subset of breast cancer see . This isWe studied a cohort of 443 patients with primary breast cancer diagnosed in the period 1988-1995. The mean length of follow-up data was 87 months (range 2 to 251 months). There were 184 breast cancer-specific deaths (mean time from diagnosis = 26 months) and 259 survivors (mean time to last follow-up date = 90 months). Primary therapy included surgical resection in all cases, followed by adjuvant hormone, radiation, and chemotherapy in 331 (75%), 160 (36%), 93 (21%) cases, respectively; 30 cases (7%) did not receive any form of systemic therapy. The clinical-pathological characteristics of the entire cohort and the Basal-like subgroup are provided (Table th percentile of the range of scores to delineate low from high expression levels. Expression of PD-L1 by tumor cells was observed in 73 (16.5%) of the 443 evaluable primary breast cancers  and showed a trend toward better OS   . Within the Basal-like subset, PD-L1 was prognostic for both RFS and OS .To validate these findings, we conducted in-silico analysis of microarray gene expression data using an online survival analysis tool to assess the prognostic effect of PD-L1 in another cohort . SupplemIn this study, we used the SP142 rabbit monoclonal antibody that recognizes an epitope in the C-terminus of human PD-L1 protein and an empirically selected cutpoint that corresponds to the upper quartile of expression levels in the cohort to determine PD-L1 tumor cell expression status and its relation to outcome in breast cancer subsets. PD-L1 was positive overall in 16.5% of a typical consecutive series of breast tumors and was associated with high TIL and with better survival outcome only in the Basal-like subtype.In assessing the literature published to date regarding tumor cell expression of PD-L1 and outcomes in breast cancer see , it is iAlthough the majority of Triple Negative breast cancers have Basal-like characteristics , these two terms are not synonymous . SeveralPD-L1 is expressed by many cell types and in the tumor microenvironment PD-L1 is predominantly expressed by CD68 macrophages . But it In conclusion, these observations are consistent with the view that PD-L1 expression in tumor cells represents an adaptive immune resistance mechanism and is associated with a better prognosis. Moreover, our results suggest that Basal-like breast cancers may respond best to PD1/PD-L1 blockade.A cohort of 443 breast cancer cases was studied representing primary tumors collected by the Manitoba Breast Tumor Bank , 46. Ki6Primary tumors were represented in tissue microarrays (TMAs) compiled by the Tumor Bank. To construct a TMA, all cases were initially selected from the database and then sections were re-reviewed to confirm and select areas for coring of corresponding blocks. Duplicate tissue cores (0.6 mm diameter) were taken from central cellular areas of each tumor with a tissue arrayer instrument . The original cohort of 636 cases was arrayed across 7 blocks. Prior utilization of these blocks and exhaustion of individual cores meant that the final interpretable cohort for this study was 443 cases.PD-L1 and CD8, CD4, CD68 staining was performed on deparaffinized sections from TMAs using a Biocare Medical Intellipath FLX autostainer using reagents from Biocare  unless otherwise noted. Slides were deparaffinized manually through xylene and graded alcohols then antigen retrieval performed in Biocare's decloaking chamber using Diva decloaking solution for 125°C for 30 seconds. Slides were loaded into the Intellipath FLX, subjected to non-specific blocking with Peroxidased-1 and background sniper then incubated with either PD-L1 , CD8 , CD4  or CD68  in Da Vinci Green diluents for 30 minutes at room temperature. The slides were then incubated with either Rabbit--HRP or Mach2 Mouse-(CD8) polymer for 30 minutes at room temperature and then detected with IP DAB for 5 minutes followed by counterstaining with a 1:10 dilution of CAT hematoxylin, air drying and coverslipping with Ecomount.th quartile. TIL markers were assessed as described previously [th percentile) of epithelial and stromal TIL- density scores (eCD8 and sCD8).IHC scoring was performed by an experienced breast pathologist (PHW) in a blinded fashion with respect to each case. TMA sections were initially assessed at low magnification to select the core with the highest density of positive cells. PD-L1 expression by tumor cells was then assessed in the area of the 0.6 mm core by the H score method whereby the expression is quantitated as the product of staining intensity (ranked from 0 to 3) and proportion of positive staining tumor cells (0 to 100%) to give an expression score range from 0 to 300. PD-L1 expression in TIL was not scored. For statistical analyses, tumor cell expression was categorized into low or high expression levels based on scores below or above the upper 75eviously  by direcp-values less than 0.05. Statistical analyses were performed using Graphpad Prism 6.0  and SPSS statistics 17 .Associations between PD-L1 expression and clinical-pathological features were evaluated using chi-square test and, where necessary, Fisher t test. Comparisons of intra-epithelial and intra-stromal TIL densities stratified by PD-L1 high/low expression were performed using Mann-Whitney test. Assessment of the correlation between PD-L1 and other immune markers was performed using a nonparametric Spearman correlation. Survival was calculated using the Kaplan-Meier method, and curves were compared with the log-rank test. Multivariate survival analyses were done using Cox regression analysis. All statistical tests were two-sided with significance established at


Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or β-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state. Current approaches to visualise brown adipose tissue (BAT) rely primarily on markers that reflect its metabolic activity. Here, the authors show that PD-L1 is expressed on brown adipocytes, does not change upon BAT activation, and that BAT volume in mice can be measured by PET-CT with a radiolabeled anti-PD-L1 antibody. PD-L1 shows broad but low expression on myeloid cells and in other tissue types, but in response to interferon-γ (IFNγ), its expression increases. A wide range of human and murine malignancies express PD-L1 constitutively or inducibly7. Despite robust expression in the tumor microenvironment and in the setting of chronic viral infections, expression of PD-L1 in naive mice is low, and mice lacking PD-L1 show only modest immunologic aberrations8.Monoclonal antibodies that target the immunological checkpoints PD-1  and PD-L1  have proven successful in the treatment of multiple cancers, notably metastatic melanoma, where PD-1 blockade is now part of the standard of care11. To this end, we developed camelid single-domain antibodies, also known as VHHs, against immune surface proteins to monitor inflammation in the tumor microenvironment by immuno-positron emission tomography-computed tomography (PET-CT)12. We sought to extend our method to imaging lower abundance immune receptors, and chose PD-L1 as both a clinically relevant target and a protein with weak expression in naive animals11. In the course of these experiments, we identified brown adipocytes as the major source of surface-disposed PD-L1 expression in naive mice.Despite the impressive gains in immunotherapy for cancer, heterogeneous outcomes necessitate new methods to monitor and predict patient responses. A method that comprehensively monitors PD-L1 expression could be of diagnostic value, and may help resolve lingering questions about the role of PD-L1 expression in checkpoint blockade responses13. In brown adipocytes, the generation of ATP from the breakdown of glucose and fatty acids is interrupted by the expression of Ucp1 (Uncoupling protein 1) in the inner mitochondrial membrane, where it dissipates the proton gradient established by the electron transport chain with concomitant release of heat14. Ucp1 expression is up-regulated by cold exposure and subsequent signaling through β-adrenoreceptors15 While it was previously believed that adult mammals lack BAT, imaging with the glucose analog 2-18F-fluorodeoxyglucose (18F-FDG) by positron emission tomography (PET) shows that adult humans have small residual BAT stores18. BAT is typically identified with functional markers that monitor BAT activity, using traceable metabolites like 18F-FDG, but there are currently no means to visualize non-activated BAT, although tissue can be identified by histological means in the absence of 18F-FDG uptake19. Unlike metabolite-based imaging reagents, the ability to visualize PD-L1 expression on brown adipocytes is independent of temperature exposure or β-adrenergic signaling, and shows robust staining of BAT deposits not visible by other non-invasive methods. Our studies thus provide a new tool to screen for therapeutic interventions of BAT function in metabolic disorders. Imaging of BAT will be essential if we are to harness its biology for the treatment of obesity, type 2 diabetes, and other metabolic disorders20.Activated brown adipose tissue (BAT) increases body temperature and energy expenditure in infants and hibernating animals23. We mapped the epitope recognized by B3 using a panel of HEK 293 derivatives transfected with constructs specifying single amino acid substitutions in PD-L1. When we mapped the mutations that abolish binding of B3 to PD-L1 onto the known structure of PD-L1, they clustered in and around the site known to interact with its ligand, programmed cell death protein 1 (PD-1)  for PD-L1 binding, with a potency similar to that of the commercial monoclonal antibody , termed B3 and A12, both of which bind specifically to PD-L1 with overlapping binding epitopes and estimated affinities in the low nM range  in wild-type (WT), but not PD-L1 knockout (KO), mice  chimeras and imaged them 7–8 weeks after transplant with radiolabeled B3 to determine whether PD-L1 was expressed on non-hematopoietic cells in BAT. At this time point, chimerism in the spleen is complete for both lymphoid and myeloid cells (data not shown). We saw a strong PET-CT signal in BAT from irradiated WT mice engrafted with PD-L1 KO bone marrow, a signal absent from PD-L1 KO recipients that received WT bone marrow Fig. . Consist27. Low levels of expression of PD-L1 and PD-1 on pre-adipocytes were evident by flow cytometry  of collagenase-digested BAT. This isolated fraction contained high levels of Ucp1 RNA when compared to floating cells isolated from subcutaneous white adipose tissue (scWAT), and was negative for hematopoietic markers when compared to the stromal vascular fraction (SVF), confirming the purity of mature brown adipocytes -18F-fluoro-6-thia-heptadecanoic acid) is linked to its metabolic activity, specifically the presence and activity of the glucose transporters responsible for uptake of 18F-FDG29. Cold exposure or treatment with β-adrenoreceptor agonists can modulate 18F-FDG uptake but cannot visualize BAT mass per se. Imaging of total BAT volume, independent of its metabolic activity has not been possible until now. This is a serious limitation, when screening for metabolic interventions considered for the treatment of obesity, type 2 diabetes and other metabolic disorders. Anti-PD-L1 imaging enables direct detection of brown adipocytes independent of their metabolic activity. To verify this, we determined whether PD-L1 levels change in brown fat in response to signals that activate BAT. We first subjected C57BL/6 WT mice to a cold shock at 4 °C for 1 h, followed by imaging with 64Cu-labeled B3. Consistent with the hypothesis that PD-L1 expression is not substantially altered by BAT activation, cold-exposed mice showed PD-L1 signals that were identical to the signal from mice housed at room temperature (25 °C)  Fig. , with a °C) Fig. ; no clea°C) Fig. . Whereas°C) Fig. , despite°C) Fig. . The add°C) Fig. . In addiThe PD-1/PD-L1 axis has attracted attention in immunotherapy of cancer, mostly because of its correlation with exhaustion of T cells and the ability to reverse such exhaustion through application of blocking antibodies. We now show that BAT is the principal tissue expressing PD-L1 in the naive mouse, with signal intensities higher than from any other tissue and comparable to murine malignancies such as the widely used B16 melanoma. A variety of cell types express PD-L1 at low levels. Many normal and malignant tissues increase PD-L1 expression in response to IFN-γ. Previous studies have speculated that PD-L1 may be expressed on adipose tissue-associated hematopoietic cells as a marker of immune cell infiltration, based largely on low-resolution imaging and ex vivo scintillation counting. Through a combination of biochemical and genetic approaches we show that brown adipocytes themselves express PD-L1. Using high resolution VHH-based anti-PD-L1 immuno-PET, we identify even small brown adipose deposits within the mouse thorax, independent of strain or age. PD-L1 should thus be considered a general marker for brown fat in preclinical mouse models, where it can be used also for longitudinal observations. In humans, brown fat deposits are generally small, and are unlikely to be properly visualized using traditional antibody scaffold-based imaging, as is suggested by the comparatively poor resolution of imaging techniques that use full-sized antibodies in the mouse.18F-FDG, a glucose analog that is taken up by metabolically active cells, including brown adipocytes. However, relying on metabolic activity as the only means of detecting brown fat has obvious limitations, particularly when aiming for modification of its activity either genetically or pharmacologically. We found that PD-L1 expression was largely independent of metabolic activity and could be used to label BAT in naive mice as well as in animals undergoing cold stress or treatment with β-agonists. Our results establish the first available method to detect and measure total brown fat volume independent of its metabolic activity. Whether PD-L1 is also expressed on beige adipocytes in mice is presently unclear, but is an important area of investigation. Given the clinical interest in PD-L1 as both a therapeutic target and a tumor biomarker, whether this molecule is also present on beige and brown adipocytes in humans is a question that needs answering. The size of human brown fat stores may not allow their visualization using currently available full-sized 89Zr conjugated antibodies for imaging, due to the poor resolution of antibody-based PET. An approach similar to that described here may need to be developed to enable visualization of human PD-L1.In live animals brown fat is imaged almost exclusively by using 32. B16 melanoma expresses both PD-1 and PD-L1, with signaling through PD-1 on tumor cells leading to enhanced tumorigenesis. When PD-1 binds to its target ligand, T cells show repressed glycolysis and enhanced lipolysis32, with a possible role for the T cell-specific costimulatory molecule CD28, although details of the signaling events that control these effects remain to be worked out. Whether the findings reported for T cells represent a wider role for PD-1/PD-L1 in metabolic regulation remains unknown. Signaling properties of PD-L1 have gone mostly unexplored. Our data not only demonstrate a clear role for PD-L1 as a marker for brown fat, but also suggest the possibility that PD-L1 has a more general metabolic function, potentially even regulating the activity of this important metabolic organ.In addition to their well-studied role in modulating TCR signaling, clues that the PD-1/PD-L1 axis also affects metabolic pathways are beginning to emergeVicugna pacos) was purchased locally, maintained in pasture, and immunized following a protocol authorized by the Tufts University Cummings Veterinary School Institutional Animal Care and Use Committee (IACUC).All mice were housed at either the Dana Farber Cancer Institute or the Whitehead Institute for Biomedical Research and were maintained according to protocols approved by the DFCI Committee on Animal Care or the MIT Committee on Animal Care, respectively. C57BL/6 and BALB/c mice were purchased from Jackson Labs or bred in house. PD-L1 knockout mice were a gift from Arlene Sharpe . A three year old male alpaca (Vicugna pacos) was immunized with a mixture of recombinant mouse and human proteins, including mouse PD-L1. A primary immunization was followed by four boosts, spaced two weeks apart, each containing 200 μg of mPD-L1 mixed 1:1 in alum (Thermo Scientific). Following immunization, total RNA was isolated from  ~ 106 fresh PBLs using the RNeasy Plus Mini Kit (Qiagen), following the manufacturer’s instructions. First strand cDNA synthesis was performed using SuperScript III reverse transcriptase (Life Technologies) and a combination of oligo dT, random hexamer or immunoglobulin-specific primers, AlCH2 and AlCH2.2, as previously described22. PCR amplification of VHH sequences and phage library generation followed a previously published procedure. Following transformation into TGI cells (Agilent), the total number of independent clones was estimated to be 3.2 × 106. Ninety-six clones were selected at random and sequenced to assess library diversity. The resulting phagemid libraries were stored at −80 °C.A male alpaca  with 50 µg/ml ampicillin. The culture was grown to mid-log phase, and infected with 100 µl 10E. coli ER2738 (NEB) for 15 min at 37 °C, followed by elution with 200 mM glycine, pH 2.2, for 10 min at room temperature. The glycine elution was neutralized and pooled with the E. coli ER2738 culture, and plated onto 2YT agar plates supplemented with 2% glucose, 5 µg/ml tetracycline and 10 µg/ml ampicillin, and grown overnight at 37 °C. A second round of panning was performed with the following modifications: 2 µg of biotinylated PD-L1-Fc was used as bait, and incubated with 2 µl 1014 PFU/ml M13 phage displaying the first-round VHH library for 15 min at 37 °C, followed by extended washes in PBS with 0.1% Tween-20.One hundred micrograms of recombinant mouse PD-L1-Fc was biotinylated by coupling Chromalink NHS-biotin reagent (Solulink) to primary amines for 90 min in 100 mM phosphate buffer, pH 7.4, 150 mM NaCl. The reaction was then run through an Amicon 10 kDa MWCO concentrator (EMD Millipore) to remove remaining NHS-biotin. Incorporation of biotin was monitored spectrophotometrically, following the manufacturer’s guidelines. 100 µl MyOne Streptavidin-T1 Dynabeads (Life Technologies) were blocked in PBS plus 2% (w/v) mouse serum in a microfuge tube for 2 h at 37 °C. Following blocking, 20 µg biotinlyated PD-L1-Fc in mouse serum was added to the beads, and incubated for 30 min at room temperature, with agitation. The beads were then washed 3× in PBS, and 200 µl of 1014 PFU/ml M13 phage displaying the VHH library were added in PBS/mouse serum for 1 h at room temperature. The beads were then washed 15× with PBS, 0.1% Tween-20. Phage was eluted by the addition of Following two rounds of phage panning, 96 colonies were isolated in 96-well round-bottom plates and grown to mid-log phase at 37 °C in 200 µl 2YT, 10 µg/ml ampicillin, 5 µg/ml tetracycline, induced with 3 mM IPTG and grown overnight at 30 °C. Plates were centrifuged at 12 X kg for 10 min, and 100 µl of supernatant was mixed with an equal volume of PBS, 5% (w/v) nonfat dry milk. This mixture was added to an ELISA plate coated with 1 µg/ml PD-L1-Fc. Following four washes in PBS, 1% Tween-20, anti-Etag antibody (Bethyl) was added at a 1:10,000 dilution in PBS, 5% (w/v) nonfat dry milk for 1 h at room temperature. The plate was developed with fast kinetic TMB (Sigma) and quenched with 1 M HCl. Absorbance at 450 nm was determined in a plate reader .E. coli periplasmic expression vector pHEN6 using the PCR primers 189 (5′-tactcgcggcccagccGGCCCAACCGGCCATGGC-3′) and 190 (5′-agtcctcctgaggagacggtgaccGAGACGGTGACCTGGGTCCCC-3′) to allow for Gibson cloning and the inclusion of a C-terminal sortase motif and 6xHis tag. WK6 E. coli containing the plasmid were grown to mid-log phase at 37 °C in TB plus ampicillin, and induced with 1 mM IPTG overnight at 30 °C. Cells were harvested by centrifugation at 5000 × g for 15 min at 4 °C, then resuspened in 25 ml 1x TES buffer  per liter culture, and incubated for 1 h at 4 °C with agitation. Resuspended cells wre then submitted to osmotic shock by diluting 1:4 in 0.25× TES, and incubating overnight at 4 °C. The periplasmic fraction was isolated by centrifugation at 8000 rpm for 30 min at 4 °C, and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole and 10% glycerol, then loaded onto a Superdex 75 10/300 column in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. Recombinant VHH purity was assessed by SDS-PAGE, and peak fractions were recovered and concentrated with an Amicon 10,000 KDa MWCO filtration unit (Millipore), and stored at −80 °C.B3 coding sequence were sub-cloned into the S. aureus Sortase A was used to label B3 and A12 by incubating 30 uM of purified VHH protein with 5uM 7 M SrtA and 100 uM GGGK-Biotin or GGGK-Alexa647 nucleophiles in 50 mM Tris, pH 8, 150 mM NaCl for 2 h at room temperature. Unreacted VHH and 7 M SrtA were removed by adsorption onto Ni-NTA agarose beads (Qiagen). The unbound fraction was concentrated and excess nucleophile with an Amicon 3,000 KDa MWCO filtration unit (Millipore), and stored at −80 °C.A heptamutant variant of High binding plates microtiter plates (Corning) were coated with recombinant Fc fusion proteins (S.A.) overnight at 25 ng/ml  or 100 ng/ml (hPD-L1) in carbonate buffer. Biotinylated VHHs or antibodies (BD Biosciences) were incubated on the coated plates in 10% inactivated fetal calf serum in phosphate buffered saline for 1 h in the presence or absence of unlabeled VHH or antibody across a range of concentrations, washed in 0.5% Tween in PBS and then developed using streptavidin-HRP (BD biosciences) and tetramethylbenzidine (TMB). For plate-bound ligand binding assays, B7-1 and PD-1 Fc fusions (R&D systems) were incubated with plate-bound ligands for 1 h in the presence of VHHs or antibodies. Plates were then washed and binding was determined using biotinylated polyclonal antibodies against B7-1 and PD-1  respectively, followed by incubation with streptavidin-HRP and then development with TMB.Cells were analyzed using a FACS Fortessa (BD) or LSR II (BD). All antibodies were directly conjugated and obtained from BD Pharmingen with the exception of antibodies to mouse and human PD-L1 (BioLegend), mouse CTLA-4 (BioLegend), mouse TIM3 (BioLegend) and mouse CD44 (Ebiosciences). All antibodies were used at a 1:100 dilution. Staining protocols followed manufacturers instructions. VHHs used in flow cytometry were directly conjugated to Alexa647, diluted to 2 µg/ml in 10% IFCS in PBS and incubated with cells for 15 min on ice. Spleen populations were identified using the following markers: B cells (CD19 + ), CD4 T cells (CD3 + CD4 + ), CD8 T cells (CD3 + CD8 + ), myeloid DCs (CD19-CD3-CD11b + CD11c + ) according to manufacturers’ recommendations . Pre-adipocytes cells were collected by sequential filtering through 70 µm and 40 µm membranes and centrifugation. Pre-adipocytes were cultured to confluence in DMEM supplemented with glutamine (Life Technologies), 5% new-born calf serum (Invitrogen), 5% fetal calf serum and pen/strep (Life Technologies), and induced to differentiate for 5 days with DMEM containing 10% fetal bovine serum (Life Technologies), 850 nM insulin (Sigma), 0.5 µM dexamethasone (Sigma), 250 µM 3-isobutyl-1-methylxanthine, phosphodiesterase inhibitor , 1 µM rosiglitazone (Sigma), 1 nM T3 (Sigma).BAT was isolated and cultured following previously described methodsMice were euthanized and BAT was surgically removed from the interscapular region of BL6 WT or PD-L1 KO mice and associated WAT was removed. Tissue was minced with scissors and then digested for 30 min at 37 C with vigorous shaking (350 rpm). After filtration (150 um mesh) and dilution in room temperature DMEM, the cell mixture was centrifuged (700 g) at room temperature. Mature brown adipocytes (floating fraction) separated from the stromal vascular fraction (SVF) on top of the solution and were carefully pipetted off using a 1 ml pipette tip with the tip cut off to allow aspiration of larger pieces. The SVF fraction at the bottom of the tube was separately analyzed and is the source of the CD45 + cells (this is unclear). Cells were stained for 10 min at room temperature with either isotype control antibody or anti-PD-L1 (10 F.9G2), washed, and resuspended after centrifugation. Both fractions were analyzed by flow cytometry. Brown adipocytes were identified by size and granularity, and by negative staining for CD45 as previously described. Cells were analyzed on an LSRFortessa (BD Biosciences).Total RNA was isolated from in vitro cultured cells at different time points of differentiation using TRizol reagent (Life Technologies/Ambion) and a miRNeasy Mini Kit (Qiagen). Three hundred nanograms were reverse transcribed using Superscript II reverse transcriptase (LifeTechnologies/Invitrogen) using 300 ng random hexamers (LifeTechnologies/Invitrogen). The cDNA was diluted 1:10 and 2.5 µl for a 96-well plate or 1 µl for a 384 well plate were used for quantitative Real-time PCR. qPCR was carried out on an ABI7900HT Fast real-time PCR system (Applied Biosystems) and analyzed using the delta delta Ct method normalized to 18S. The following primer sets were used: 18S fwd 5′-GTAACCCGTTGAACCCCATT-3′; 18S rev 5′-CCATCCAATCGGTAGTAGC-3′; Ucp1 fwd 5′-ACTGCCACACCTCCAGTCATT-3′;Ucp1 rev 5′-CTTTGCCTCACTCAGGATTGG-3′; Pd-l1 fwd 5′-TGCTGCATAATCAGCTACGG-3′; Pd-l1 rev 5′-CCACGGAAATTCTCTGGTTG-3′.Adult WT mice were injected IP with 50 ug of B3-Alexa647 or 96G3m-Alexa647 (control VHH). Mice were sacrificed one hour after injection and interscapular BAT was collected, mounted on a coverslip and imaged with a Zeiss AxioPlan 2 microscope. Images were analyzed using Metamorph and ImageJ software.To label purified VHH with tetrazine, reactions were performed in 50 mM Tris·HCl, pH 7.5 supplemented with 10 mM CaCl2, 150 mM NaCl, 500uM triglycine-containing probe, 100 μM VHH and 5 μM sortase. After incubation at 4 °C with agitation for 2 h, reaction products were analyzed by LC-MS, with yields generally > 90%. Ni-NTA beads were added to the reaction mixture with agitation for 5 min at 25 °C, followed by centrifugation to remove sortase and any remaining unreacted His-tagged substrate. The final product was purified by size-exclusion chromatography in PBS. VHH-Tz , PBS (300 μl), and 18F-TCO in DMSO (4.0 mCi (148.0 MBq), 100 μl) were mixed in a microfuge tube. The tube was sealed and shaken at room temperature for 20 min. The mixture was analyzed by radio-TLC (instant thin layer chromatography (ITLC), 100% MeCN, Rf 18F-TCO = 0.9, Rf 18F-VHH = 0.0) showing 90% conversion to 18F-VHH. The reaction mixture was loaded onto a PD-10 size-exclusion cartridge , and eluted with PBS. Imaging was performed on a Siemens Inveon PET/CT imager. All PET/CT imaging procedures were approved by the Massachusetts General Hospital subcommittee on research animal care.g, 10 min), 15 ml 1 M MES pH 6.5 and AEBSF  was added, and the supernatant swirled at 4˚C for 1 h with a 30 ml bed volume (bv) of His60 Ni-IDA resin (Clontech). Resin was poured into a gravity column, washed with 10 bv wash buffer ) and eluted with 5bv elution buffer (wash buffer with 100 mM Arginine-Cl and 0.5 M imidazole). Protein was concentrated using centrifugal units , and applied to a gel filtration column  equilibrated in 25 mM MES, pH 6.5, 150 mM NaCl, 10% glycerol, 100 mM Arg-Cl. Peak fractions were collected and concentrated by as above.Coding sequences were cloned into a modified version of the Daedalus lentiviral transfer vector, which supported ligation independent cloning and expressed targets as a fusion with a variant of mIgG2a. Lentivirus production was essentially as described. Transduction of HEK-293F cells (Gibco) was carried out at a scale of 30 ml in Freestyle 293 Expression media (Gibco) and cultures were expanded to a 3 l production scale. Supernatant from 3 l culture was harvested by centrifugation (2000 × Murine PD-L1 was cloned into the SacI and BamHI sites of the Clontech N1 mCherry-fusion vector replacing the native leader peptide sequence with that of erythropoietin (EPO). Site-specific mutagenesis was performed using KOD polymerase, 2 mM dNTPs and 4 mM MgCl2. Solvent accessible positions for mutagenesis were selected based on the crystal structure of the complex formed by human PD-L1 and PD-1 (PDB: 3BIK) and determining the equivalent positions in mouse PD-L1 using Custal2 sequence alignment. Each chosen position was mutated to an Ala, Glu or Arg residue with an overall success rate of  ~ 70%. All sequence-validated mutants were tested by transient expression in suspension HEK 293 cells prior to use in the nanobody epitope mapping experiments.g for 5 min, the supernantant removed and 200 μl of 1× PBS and 0.2% BSA was added to wash. After two additional washes, cells were resuspended in 100 μl and analyzed using an Hypercyte autosampler (Intellicyt) connected to a custom Blue-Yellow laser BD Accuri flow cytometer. Counts were normalized to wild type.Wild-type and mutant PD-L1 mCherry-fusion constructs were transiently transfected into HEK 293 S cells plated in 24-well tissue culture plates using 1 ml of Freestyle media (Invitrogen) and PEI transfection. Three days post transfection, 12 wells were selected at random and counted, and the average count used to dilute cells to ~ 1 × 106 cells/ml in PBS. The Alexa 488-labeled α-PD-L1 VHH was diluted to 0.01 μg/μl in 1X PBS and 0.2% BSA. Binding reactions were setup in 96-well V-bottom plates using 150 μl cells  and 50 μl of diluted nanobody. Reactions were covered in foil and incubated at room temperature with shaking at 900 rpm in a 96-well plate shaker for 1 h. After incubation, plates were centrifuged at 500 × t-test with pooled variance if there was no evidence of inhomogeneity of variances between groups. If the variances were unequal, the exact Wilcoxon rank sum test, a non-parametric alternative to the t-test, was used. Every effort was made to keep testing consistent across related experiments. For comparisons of more than two groups, analysis of variance (ANOVA) was used if there was no evidence of inhomogeneity of variance; the Kruskal-Wallis test was the non-parametric alternative. Tumor growth studies were analyzed using mixed model ANOVA. Sample size was determined based on a review of similar experiments in the literature or based on prior experience in the lab. Animals were all randomized to treatment groups and the start of the experiment. The experiments were not blinded unless stated in the methods.Two sample comparisons used the The data sets generated and analyzed during current study are available from the corresponding author on reasonable request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5