Intracellular nucleic acid sensors often undergo sophisticated modifications that are critical for the regulation of antimicrobial responses. Upon recognition of DNA, the cytosolic sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the second messenger cGAMP, which subsequently initiates downstream signaling to induce interferon-αβ (IFNαβ) production. Here we report that TRIM56 E3 ligase-induced monoubiquitination of cGAS is important for cytosolic DNA sensing and IFNαβ production to induce anti-DNA viral immunity. TRIM56 induces the Lys335 monoubiquitination of cGAS, resulting in a marked increase of its dimerization, DNA-binding activity, and cGAMP production. Consequently, TRIM56-deficient cells are defective in cGAS-mediated IFNαβ production upon herpes simplex virus-1 (HSV-1) infection. Furthermore, TRIM56-deficient mice show impaired IFNαβ production and high susceptibility to lethal HSV-1 infection but not to influenza A virus infection. This adds TRIM56 as a crucial component of the cytosolic DNA sensing pathway that induces anti-DNA viral innate immunity. The protein cGAS responds to the presence of cytosolic DNA by producing the second messenger cGAMP, which triggers antiviral interferon responses. Here, Seo et al. show that ubiquitination by the E3 ligase TRIM56 enhances cGAS activity and is important for the immune response against DNA viruses. Microbe-derived nucleic acids are potent PAMPs that elicit PRR-mediated host immune responses3. The recognition of invading RNA viruses by cytoplasmic sensors (RIG-I and MDA5) and endosomal toll-like receptors (TLRs) has been extensively characterized3. The appearance of naked DNA in the cytoplasm of mammalian cells also triggers DNA sensor-mediated signal transduction4. Many cytosolic DNA sensors have been identified, including AIM27, DAI8, DDX419, DNA-PK10, IFI1611, a form of RNA polymerase III13, and cyclic GMP-AMP (cGAMP) synthase (cGAS)15. Particularly, recent studies have shown that cGAS functions as the primary cytosolic DNA sensor that triggers production of type I interferons (IFNs) and other inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, upon DNA transfection and DNA virus infection15. Following activation, cGAS converts ATP and GTP into the dinucleotide cGAMP18. cGAMP is a second messenger that binds to stimulator of interferon genes (STING), which in turn induces the recruitment of TANK-binding kinase 1 (TBK1) and interferon regulatory factor-3 (IRF-3)19. Then, the TBK-mediated activation of the IRF-3 pathway induces the expression of type I IFNs. Thus cGAS-mediated DNA sensing signals through various adaptor molecules to ultimately induce potent antiviral innate immunity.Pathogens are detected by host pattern recognition receptors (PRRs) that sense various microbial motifs collectively known as pathogen-associated molecular patterns (PAMPs) and subsequently elicit antimicrobial innate immune responses20. Given the central role of the cGAS pathway in the innate immune response to viral infections, it is expected that various modulations and modifications to cGAS control its activity. We have previously reported that the autophagy protein Beclin-1 negatively regulates cGAS activity21. Beclin-1 directly interacts with cGAS and suppresses cGAMP synthesis and signaling. On the other hand, this interaction enhances the autophagy-mediated degradation of cytosolic pathogen DNA to avoid persistent immune stimulation. Several posttranslational modifications, including phosphorylation and glutamylation, have been reported to play critical roles in regulating the cGAS-STING pathway22. Glutamylation of cGAS impairs its DNA binding and enzymatic activity23. We have also shown that Akt kinase suppresses cGAS enzymatic activity by phosphorylating its carboxyl-terminal enzymatic domain. This suppresses the subsequent antiviral cytokine production and leads to increased DNA virus replication24. These modulations fine-tune the IFN-mediated antiviral pathway to ultimately ensure that the host-DNA-sensing innate immune response is kept in balance after responding to stimuli, such as DNA virus infections.Since both self and non-self DNA can activate intracellular DNA sensors, this DNA sensing pathway must be tightly regulated to prevent harmful activity arising from unrestrained signaling25. The interaction between endoplasmic reticulum ubiquitin ligase RNF185 and cGAS specifically catalyzes the K27-linked polyubiquitination of cGAS, which promotes its enzymatic activity26. Members of the tripartite motif (TRIM) E3 ubiquitin ligase family have arisen as key molecules in antiviral immunity, either as direct restriction factors of viral replication or as regulators of nucleic acid sensing pathways27. TRIM25 and TRIM4 mediate the K63-linked ubiquitination that activates RIG-I cytosolic RNA sensor28. On the other hand, TRIM14 inhibits the degradation of cGAS DNA sensor mediated by selective autophagy receptor p62, which promotes innate immune responses. TRIM56 has been shown to be a restriction factor of several RNA viruses  both in an E3 ligase-dependent and -independent manner31. Furthermore, Salmonella typhimurium SopA HECT-type E3 ligase targets TRIM56 and TRIM65 to stimulate RIG-I and MDA5 innate immune receptors, which subsequently modulates inflammatory responses32. However, since previous studies were limited to in vitro cell culture assays, the in vivo role of TRIM56 still remains unclear. While an initial report described a direct role of TRIM56 in the STING-mediated double-stranded DNA sensing pathway, a later study convincingly disputed that TRIM56 has no role in the STING-mediated pathway, suggesting that alternative mechanisms or functions of TRIM56 should be explored34.Protein ubiquitination controls a large number of cellular processes, including protein degradation, DNA repair, chromatin remodeling, cell-cycle regulation, endocytosis, kinase signal pathways, and othersIn order to determine the specific in vivo role of TRIM56, we generated TRIM56-deficient cells and mice and identify that TRIM56 directly targets cGAS, rather than STING or its downstream signaling molecules, to confer DNA sensing-mediated innate immune responses. TRIM56 interacts with the amino-terminal regulatory domain of cGAS and this interaction promotes the Lys335 monoubiquitination of cGAS, resulting in the increase of its cGAMP production. Consequently, TRIM56 deficiency leads to a dramatic reduction of cGAS-mediated IFNαβ production upon herpes simplex virus type 1 (HSV-1) infection but not upon influenza A virus (IAV) infection. Our in vitro and in vivo study indicates that the TRIM56-induced monoubiquitination of cGAS is important for efficient cytosolic DNA sensing in response to DNA virus infection.35. Specifically, this approach immobilizes protein complexes from lysed cells directly on a coverslip, which is then studied under a total internal reflection fluorescence microscope. This SiMPull assay also revealed that TRIM56 effectively bound to cGAS  cGAS fused with maltose-binding protein (MBP) interacted with TRIM56. However, both MBP-cGAS C-terminal region (a.a. 161–522) and MBP alone failed to interact with TRIM56  to deplete TRIM56 expression  DNA transfection or cGAMP stimulation. Depletion of TRIM56 expression led to the significant reduction of HT-DNA mediated IFNβ mRNA expression but did not affect cGAMP-mediated IFNβ mRNA expression  cells36 and complemented with mouse cGAS-FLAG  cGAS(−), TRIM56(−)cGAS(+), and TRIM56(−)cGAS(−) cells failed to induce IFNβ mRNA expression upon HT-DNA transfection, whereas these cells effectively induced IFNβ mRNA expression upon cGAMP stimulation  cGAS(+) L929 cells were capable of inducing IFNβ mRNA expression upon either HT-DNA or cGAMP stimulation  cGAS(−), TRIM56(−) cGAS(+), and TRIM56(−) cGAS(−) cells failed to induce IP-10 mRNA expression upon HSV-1ΔICP34.5 infection, whereas wild-type (WT) L929 cells were capable of inducing IP-10 mRNA expression upon HSV-1ΔICP34.5 infection  cGAS(−), TRIM56(−) cGAS(+), or TRIM56(−) cGAS(−) L929 cells were stably transfected with an ISRE-IFNβ promoter-driven secreted luciferase reporter, followed by stimulation with HT-DNA, pdAdT, pdGdC, pI:C, or cGAMP. TRIM56(+) cGAS(−), TRIM56(−) cGAS(+), or TRIM56(−) cGAS(−) cells also failed to induce ISRE-IFNβ promoter-luciferase expression upon HT-DNA, pdA:dT, or pdG:dC stimulation, but they effectively induced ISRE-luciferase expression upon pI:C or cGAMP stimulation  cGAS(−), TRIM56(−) cGAS(+), or TRIM56(−) cGAS(−) L929 cells showed defective ISRE-IFNβ promoter-luciferase expression under the same conditions  cGAS(−), TRIM56(−) cGAS(+), or TRIM56(−) cGAS(−) cell lysates were added into digitonin-permeabilized THP1-Lucia™ ISG reporter cells to perform a cGAMP bio-assay  cGAS(−), TRIM56(−) cGAS(+), or TRIM56(−) cGAS(−) cell lysates showed little or no increase of cGAMP-mediated IFN responsiveness -cGAS effectively co-localized at the indicated foci upon HT-DNA stimulation or HSV-1 infection but not upon SeV infection. However, STING did not co-localize with either cGAS or TRIM56 under any conditions  cGAS(−) or TRIM56(−) cGAS(−) L929 cells, and purified cGAS-FLAG protein was then subjected to immunoblotting with an anti-VU-1 antibody. This experiment showed that monoubiquitination of cGAS-FLAG could be only detected in TRIM56-expressing cells  of cGAS that are targeted by TRIM56 for monoubiquitination, cGAS-FLAG was co-expressed with TRIM56 in HEK293T cells and partially purified for multi-dimensional liquid chromatography coupled with tandem mass spectrometry  L929 cells were stably complemented with cGAS WT or the K335R mutant followed by HT-DNA stimulation or HSV-1ΔICP34.5 infection. cGAS WT expression in cGAS(−) L929 cells robustly induced IFNβ mRNA production upon HSV-1ΔICP34.5 infection or HT-DNA stimulation, whereas expression of the cGAS K335R mutant led to a minimal induction of IFNβ mRNA under the same conditions -binding activity of cGAS WT but not the cGAS K335R mutant  rapidly lost weight, showed severe disease symptoms, and died over a period of 5 days  showed minor symptoms, recovered their lost weight, and survived; 33% of them (n = 2) showed moderate symptoms and died over a period of 6–7 days postinfection  showed nearly no difference from WT mice (n = 9) in response to in vivo IAV PR8 infection; both mice rapidly lost weight and died over a period of 11 days  were infected with HSV-1 or HSV-1ΔICP34.5  of TRIM56 by replacing the cysteine 21 and 24 residues with serines and complemented TRIM56−/− BMDMs with lentivirus-derived TRIM56WT or TRIM56 Mut  and bone marrow-derived dendritic cells (BMDCs). Similarly, we observed that TRIM56−/− MEFs failed to induce IFNβ production following HT-DNA or pdA:dT stimulation compared to WT MEFs , Sindbis virus, or Parainfluenza virus  were housed in specific pathogen-free barrier facilities. Male and female mice aged 6–14 weeks were used. Primary bone marrow and peritoneal cavity cells were collected from WT and TRIM56 knockout mice for BMDMs and BMDCs. All experiments were approved and done according to the guidelines of the Institutional Animal Care and Use Committee at the University of Southern California. Sample sizes were estimated based on previous similar studies for innate immunity. Animal experiments were not randomized or blinded.36, and Vero cells  were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium  containing 4 mm glutamine and 10% fetal bovine serum (FBS). HEK293T, L929, and Vero cells are authenticated by ATCC and were not further validated in our laboratory. Transient transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cell lines stably expressing cGAS were generated using a standard selection protocol with 2 µg/ml of puromycin (Invitrogen). Target sequences to generate stable cell lines are presented in Supplementary Table The reagents were purchased from the following companies: HT-DNA and anti-FLAGM2-agarose (Sigma); pdAdT, pdGdC, pI:C (Invivogen); and cGAMP . To clone mouse TRIM56, we purchased an expressing plasmid (Addgene). Mouse TRIM56-V5 constructs were created by subcloning the PCR products into pEF-IRES-puro. Mutagenesis by overlap extension PCR was used to create mouse TRIM56 Mut, which was subcloned into pEF-IRES-puro. Primers used for cloning are presented in Supplementary Table 50.HEK293T cells were collected 48 h after transfection with cGAS-FLAG and/or TRIM56-V5 and lysed with NP-40 buffer  supplemented with complete protease inhibitor cocktail (Roche). Post centrifugation, supernatants were mixed with a 50% slurry of FLAG M2 beads , and the binding reaction mix was incubated for 4 h at 4 °C. Precipitates were washed extensively with lysis buffer. Proteins bound to FLAG M2 beads were eluted and separated in a NuPAGE 4–12% Bis-Tris gradient gel (Invitrogen). After Coomassie brilliant blue staining, protein bands corresponding to the cGAS-FLAG were excised and separately analyzed by ion-trap mass spectrometry at the Harvard Taplin Biological Mass Spectrometry Facility in Boston, MAPolyethylene glycol (PEG)-coated quartz slides with flow chambers were obtained according to previous protocols. A PEG surface was coated with Neutravidin (0.05 mg/ml) followed by anti-GFP or V5  antibody conjugated with biotin (Rockland) and incubated for another 5 min in T50 (10 mM Tris pH 8 and 50 mM NaCl). Enhanced GFP-fused cGAS cell lysates were added to the antibody-coated surface and incubated for 5–10 min. The exposure time was 30 ms and the single-molecule signals were processed with a custom-edited IDL and Matlab program.HEK293T cells were seeded into 12-well plates. Twenty-four hours later, the cells were transfected with 100 ng of IFNβ luciferase reporter plasmid and 20 ng of TK-renilla luciferase. In addition, 100 ng of plasmid encoding human or mouse cGAS, 50 ng of plasmid encoding STING, or 400 ng TRIM56 was transfected. Forty-eight hours after transfection, whole-cell lysates were prepared and subjected to the Dual-Glo luciferase assay according to the manufacturer’s instructions (Promega). Results are presented with Renilla luciferase levels normalized by the firefly luciferase levels.Cell lysates were collected in 1% NP-40 buffer and quantified by BCA protein assay (Thermo Scientific). Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Bio-Rad) by semi-dry transfer at 25 V for 40 min. All membranes were blocked in 5% milk in Tris-Buffered Saline (TBST) and probed overnight with the indicated antibodies, which were diluted in the blocking buffer solution at 4 °C. Primary antibodies included mouse FLAG , rabbit V5 , and mouse VU-1 . Horseradish peroxidase-conjugated secondary antibodies were incubated on membranes in 5% milk in TBST, and bands were developed with ECL reagent (Thermo Scientific) and imaged using a Fuji LAS-4000 imager.For immunoprecipitation, cells were harvested and then lysed in 1% NP-40 buffer supplemented with a complete protease inhibitor cocktail (Roche). After preclearing with protein A/G agarose beads for 1 h at 4 °C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies. A 50% slurry of FLAGM2 beads  was added to 1 ml of cell lysates and incubated at 4 °C for 4 h. Immunoprecipitates were extensively washed five times with lysis buffer and eluted with SDS loading buffer by boiling for 5 min. Full-length uncropped blots are presented in Supplementary Fig. Escherichia coli were grown at 37 °C until reaching OD600 = 0.6. The temperature was then shifted to 18 °C and grown overnight by adding 1 mM IPTG. Cells were collected by centrifugation and lysed with 1% NP40 buffer. Clarified lysates were mixed with Ni-NTA agarose (Invitrogen) and washed five times with lysis buffer prior to elution of protein using 150 mM imidazole. The concentrated protein was aliquoted and stored at −80 °C for the in vitro cGAS enzyme assay and binding assay.MBP-cGAS (mouse) fusion protein (151–522 AA) and full length were expressed in BL21 . 2+ATP, EDTA (50 mM), 20 × E1, 10 × E2, TRIM56 E3, cGAS, 20xBt-Ub) were added to 1.5 ml Eppendorf tubes and the tube contents were mixed gently. The mixture was incubated at 37 °C for 60 min. The assay was quenched by addition of 50 μl 2× non-reducing gel loading buffer. It proceeded directly to western blot.The in vitro ubiquitination assays using TRIM56 (E3) and MBP-cGAS as substrates were performed using a non-radioactive assay (Enzo Life Sciences). Briefly, assay components , dithiothreitol (DTT) (50 mM), Mg32 alpha phosphate radiolabed ATP in buffer . After a 1 h incubation at 37 °C, the reaction was treated with 5 units of alkaline phosphatase (Roche) for 30 min to stop the reaction and 1 μl of reaction solution was spotted onto TLC plates  with the solvent 1:1.5 [v/v] 1 M (NH4)2SO4 and 1.5 M KH2PO4. To visualize the reaction, the TLC plates were air-dried and imaged using a Fuji Phosphor Imager.For in vitro cGAS enzymatic reactions, 1 μM MBP-cGAS or MBP-cGAS variant was mixed with 250 μM GTP and 10 μCi PTotal RNA was isolated from cells with TriReagent (Sigma). cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad), and quantitative PCR reaction was monitored with SYBR Green Supermix (Bio-Rad). Primers used for real-time PCR are presented in Supplementary Table L929 cells were treated with HT-DNA. The cell culture supernatant was collected and measured for IFNα or IFNβ by sandwich ELISA using a mouse IFNα or IFNβ ELISA Kit  according to the manufacturer’s protocols.HSV-1 strain 17+ were grown in Vero cells propagated in DMEM supplemented with 10% FBS and antibiotics. BMDM cells were inoculated with virus in DMEM supplemented with 2% FBS for 1 h. After incubation for the indicated times , culture medium was harvested and the viral titer was determined. Briefly, culture medium from BMDM cells was serially diluted in DMEM supplemented with 10% FBS and used to infect 80% confluent monolayers of Vero cells growing in six-well plates. After infection, the monolayers were rinsed and overlaid with 7% methylcellulose. Plaques were counted after 3–5 days.t-test with a minimum of n = 3. p-Values < 0.05 were considered significant.All data were analyzed using the Prism 5 software . All data were analyzed using a two-tailed Student’s The data that support the findings of this study are available in this article and its Supplementary Information files or from the corresponding author upon request.Supplementary InformationPeer Review File


The human LINE-1 (or L1) element is a non-LTR retrotransposon that is mobilized through an RNA intermediate by an L1-encoded reverse transcriptase and other L1-encoded proteins. L1 elements remain actively mobile today and continue to mutagenize human genomes. Importantly, when new insertions disrupt gene function, they can cause diseases. Historically, L1s were thought to be active in the germline but silenced in adult somatic tissues. However, recent studies now show that L1 is active in at least some somatic tissues, including epithelial cancers. In this review, we provide an overview of these recent developments, and examine evidence that somatic L1 retrotransposition can initiate and drive tumorigenesis in humans. Recent studies have: (i) cataloged somatic L1 activity in many epithelial tumor types; (ii) identified specific full-length L1 source elements that give rise to somatic L1 insertions; and (iii) determined that L1 promoter hypomethylation likely plays an early role in the derepression of L1s in somatic tissues. A central challenge moving forward is to determine the extent to which L1 driver mutations can promote tumor initiation, evolution, and metastasis in humans. Transposable genetic elements constitute at least 45% of the human genome . Some ofsequence ,4. The eIn order to examine how L1s influence human genomes and disease, we must first understand how these elements are mobilized .cis preference for mobilizing the specific mRNA that gave rise to them, which allows for the preferential amplification of functional L1 copies [This process begins with a canonical full-length L1 (FL-L1) source element that is (A) tail A. Only apromoter A, most litiation . ORF2p iitiation  and reveitiation . These pitiation ,11 that 1 copies ,13. Receng genes . Althougng genes .Bombyx mori [The final step of L1 mobilization is known as target-primed reverse transcription (TPRT) F. This mbyx mori , and latbyx mori . BrieflyTTT/A-3′ ,17,18, aTTT/A-3′ . This cl(A) tail ,21 and ethe mRNA ,16. Finathe mRNA .Alu [New L1 insertions have important hallmark features of retrotransposition: a poly(A) tail, flanking target site duplications (TSDs), frequent 5’ truncation, and occasional 5’ inversion ,24,25,26Alu  and SVA Alu ,32, as wAlu . As a coAlu ,34.The large number of individual steps that are required for L1 mobilization present numerous opportunities for human cells to regulate this mutagenic activity, and many such L1 regulators have evolved. In fact, there are mechanisms of L1 repression that act at each step of the mobilization process. For example, one of the most well-studied mechanisms of L1 repression is the methylation of CpG dinucleotides in the L1 promoter ,36. PromThe Selfish Gene in 1976, and there were even ongoing reviews and debates about this notion in Nature in 1980 [Mobile genetic elements were first discovered in maize genomes by Barbara McClintock in 1950 . This wo in 1980 ,45,46. H in 1980  and by oDuring the 1980s there was a furious rush to describe the newly-discovered L1 repetitive elements. This included classifying L1s, determining their function, and beginning to understand their activity—all of which occurred in the short span of eight years. When L1s were first discovered in 1980, they were simply described as long repeated sequences of variable length that were dispersed across all human chromosomes . ResearcF8) on Chromosome X. Two unrelated patients were found to have germline L1 insertions in the F8 gene that were absent from their parents’ genomes, indicating that these L1 insertions probably occurred in gametes or during early embryogenesis [MYC proto-oncogene. However, only part of this L1 insertion was sequenced and the structure lacked the hallmarks of TPRT [The first evidence for ongoing mobility of L1s in human genomes came in 1988, when Kazazian et al. found two disease-causing de novo germline L1 insertions . This L1ogenesis . The twoogenesis . This 7– of TPRT . Thus, aF8 [APC tumor suppressor gene (TSG) in tumors of CRC patients. This screen was performed because APC plays a pivotal role in this kind of cancer: the majority of CRC tumors are initiated by mutation or loss of function of both APC alleles in normal colon cells, which results in the formation of a precancerous lesion that can eventually progress to carcinoma [APC, so it was expected to disrupt normal APC function. The authors emphasized that the insertion likely initiated the tumor, but in view of our current understanding of CRC, this interpretation is somewhat unclear  . SimilarF8 , this inarcinoma ,54,55. TAlu, SVA, or other mRNAs (reviewed in [For many years this was the only available evidence that L1 elements were capable of retrotransposition in somatic tissues. There was another putative example of somatic L1 activity published in 1997 . Howeveriewed in ). In mosAlu [At this point in time scientists were undertaking important mechanistic research on L1 retrotransposons, including studies aimed at understanding how L1s are mobilized .As outlined above in As mentioned above, we rediscovered somatic L1 retrotransposition in human tumors in a study that leveraged next-generation sequencing technologies to investigate L1 retrotransposition in human populations and cancers . This stAfter developing and optimizing the L1-Seq approach, we used it to discover new L1 insertions in 38 diverse humans, eight tumor-derived cell lines, 20 non-small cell lung tumors , and 10 brain tumors (with matched blood leukocyte controls) . This scDuring the seven years that have elapsed since our 2010 study , many adAlthough these studies employed several strategies to measure somatic L1 activity, most of the methods can be grouped into two basic categories: (1) targeted resequencing assays and (2) bioinformatics tools that use whole genome sequencing (WGS) or whole exome sequencing (WES) to discover somatic L1 insertions. Targeted resequencing tools exploit specific sequence features of young L1s to selectively amplify and sequence novel L1 insertion junctions. These assays are similar to the previously described L1 Display  and L1-SSomatic L1 activity in cancer genomes has been found to be almost entirely confined to tumors arising from epithelial tissues  see Tab. A similer tumor .Additionally, L1s are responsible for the occasional somatic retrotransposition of other sequences in cancer genomes. The most commonly mobilized non-L1 sequences are 3ized see . In one 24% 655/26 of the 24% 655/26 of the n tumors ,88. Thes24% 655/26 of the Alu insertions also have been observed in various epithelial cancers [Processed pseudogenes also are generated in somatic tissues via the retrotransposition of cellular mRNAs by the L1 machinery ,90. Such cancers ,71,74,75It is increasingly becoming possible to identify the specific FL-L1 source elements that produce somatic offspring insertions in tumors. A key development in this regard has been a much more extensive knowledge of the FL-L1 source elements that are harbored by human genomes. Based on the reference (REF) human genome sequence, Brouha et al. initially estimated that every individual has a collection of approximately 80 to 100 FL-L1 source elements that are retrotransposition-competent . Later sCurrently, there are two available methods to determine source/offspring relationships between L1 elements. The first uses 3ductions ,73,75. Wductions . Each soductions .APC TSG and initiated tumorigenesis [Both of these methods have been used to study the FL-L1 elements that are active in human tumors. One large study employed the 312 types . Even moductions . Activitductions ,75. Thise of CRC . Three nigenesis .These studies have uncovered some important features of active source elements. For instance, both REF and non-REF FL-L1 source elements can contribute to somatic L1 retrotransposition ,80. SourThe discovery of frequent somatic L1 activity in cancer has researchers asking why this phenomenon is occurring and why it is so variable between tumors and tissues. Promoter methylation is one of the earliest lines of defense against L1 activity, and therefore, hypomethylation is thought to be a necessary step for L1 reactivation in tumors. This hypothesis has been corroborated by many groups. These studies examined methylation at three different levels, using techniques that vary in scope. First, the Illumina Infinium platform assesses global genomic methylation within CpG islands . Second, primer) ,80.Using the first method (the Illumina Infinium platform), we identified a hypomethylation signature at 59 genomic CpG sites that was associated with L1-permissive lung cancers . Soon thIn addition to cancer genomes, somatic retrotransposition also occurs in neuronal cells ,85 and nAPC TSG and likely was a driver of tumorigenesis [APC allele was not examined in that study and the authors did not rule out a possible role for faulty DNA repair as an initiator of tumorigenesis [PTEN . It canscovered ,75.One major unresolved question is: how often do L1 driver mutations initiate tumorigenesis in normal cells? Insertions that occur after tumor growth is well underway would not appear to participate in tumorigenesis per se, but instead might play a role in tumor evolution or metastasis. Doucet-O’Hare et al. examined the evolution of esophageal cancer from a precancerous condition and demonstrated that L1s can be active very early during the tumorigenesis process, and even found somatic L1 insertions in precancerous lesions that never progressed to cancer (over a 15 year period) . In a siAPC is a gatekeeper TSG that is frequently mutated in patients with CRC—in fact, both copies of APC must be mutated to initiate most cases of CRC [APC allele was inactivated by a point mutation, and that this tumor had stable microsatellites, indicating that faulty DNA repair did not initiate tumorigenesis in this case [APC gatekeeper alleles are mutated in a normal cell [APC alleles and a stop codon disrupted the second allele. Thus, somatic L1 mutations can initiate tumorigenesis in normal colon cells.We recently demonstrated that L1 indeed can initiate tumorigenesis in normal colon cells . APC is s of CRC ,54,55. Ws of CRC , only 38s of CRC  . Even cancer ).Another confounding factor is that the temporal order in which gene mutations occur during tumor initiation and evolution is unclear in most tumor types (recently reviewed in ). In manAPC occurred at the earliest stages of tumorigenesis , providing further evidence that normal colon tissues can support somatic L1 activity [Although L1 clearly is quite active in the normal somatic tissues of the brain and epithelial cancers, we are just beginning to explore the extent of somatic activity in other normal tissues. Several lines of evidence suggest that L1 can evade somatic repression in at least some other normal tissues. In one study, 21 putative somatic L1 insertions were identified in normal liver cells using a targeted sequencing assay . In anotactivity . Thus, tOn the basis of this limited evidence, is it possible that most  normal epithelial tissues support L1 activity? If so, this might have been largely missed for the same reason that it was initially overlooked in the brain: that each cell in a given tissue generates a unique collection of somatic L1 retrotransposition events that cannot be detected in bulk tissue. The solution to this problem is to adapt L1 discovery methods to single cell sequencing technologies. This approach has been pioneered in brain tissues by the Walsh  and FaulFinally, we need to address how differences in FL-L1 source element profiles influence tumorigenesis. As discussed in We also need to gain a better understanding of how FL-L1 source element profiles vary in human populations. Although some FL-L1s are ubiquitously found in most or all human genomes, many others are found only in a subset of individuals and are inherited in a population-specific manner . As a reWe have presented a brief overview of research examining somatic L1 retrotransposition in human genomes, focusing on landmark studies outlining the activity of these mobile elements in human cancers. Researchers in this field have characterized many aspects of somatic L1 activity in the short span of only seven years. However, several major questions remain unresolved. One unsettled question is: How often does somatic L1 retrotransposition initiate and drive tumorigenesis in humans? Despite the fact that thousands of somatic L1 insertions have been recovered from many tumor types, only a handful of clearly-recognizable driver mutations have been discovered. We also have a very incomplete understanding of L1 activity in normal somatic tissues. If L1s are highly active in most adult somatic tissues, we clearly need to gain a better understanding of the consequences of this activity. Likewise, in our current era of human genomics and precision medicine, it is increasingly important to define inter-individual variation and determine how variation in FL-L1 source element profiles may impact human health and disease.


The exceptional properties of graphene make it a promising candidate in the development of next‐generation electronic, optoelectronic, photonic and photovoltaic devices. A holy grail in graphene research is the synthesis of large‐sized single‐crystal graphene, in which the absence of grain boundaries guarantees its excellent intrinsic properties and high performance in the devices. Nowadays, most attention has been drawn to the suppression of nucleation density by using low feeding gas during the growth process to allow only one nucleus to grow with enough space. However, because the nucleation is a random event and new nuclei are likely to form in the very long growth process, it is difficult to achieve industrial‐level wafer‐scale or beyond (e.g. 30 cm in diameter) single‐crystal graphene. Another possible way to obtain large single‐crystal graphene is to realize ultrafast growth, where once a nucleus forms, it grows up so quickly before new nuclei form. Therefore ultrafast growth provides a new direction for the synthesis of large single‐crystal graphene, and is also of great significance to realize large‐scale production of graphene films (fast growth is more time‐efficient and cost‐effective), which is likely to accelerate various graphene applications in industry. Pt is one of the promising substrates, while at the moment it still cannot meet the demands for the large‐scale synthesis of graphene. Ni and Cu are much cheaper and widely available substrates that make the industrialized production of graphene feasible. High solubility of carbon in Ni leads to the formation of multilayer graphene.Graphene, a two‐dimensional material with carbon atoms bonded in a honeycomb lattice, has attracted immerse interests in the past decade due to its exceptional properties and various applications.However, as‐synthesized graphene films on Cu foils are typically polycrystalline and the grain boundaries degrade the properties greatly, limiting its high‐end applications.2Figure−1. That means to grow 12‐inch single‐crystal graphene films typically needs almost one day. This time‐ and energy‐consuming process is not suitable for the large‐scale production. Only from 2016, several breakthroughs made the growth rate exceed 100 µm min−1,−1.FigureAfter about a decade's efforts, the domain size of single‐crystal graphene has increased more than four orders of magnitude, from micrometres to inches Figure2.31, 334 makes it a preferred carbon source to prevent pyrolysis at high temperatures, which is favourable to surface‐mediate growth of high‐quality graphene.4 onto Cu surfaces; (ii) catalytic dissociation of CH4 by Cu, resulting in C species CHx (x = 0–3); (iii) surface diffusion of C species; (iv) C attachment onto graphene domain edges. A large number of experiments have shown that these four processes in graphene growth are dominated by the catalytic substrate, feeding gas, reaction barriers, temperature and pressure  that is catalytically decomposed from CH4 and absorbed on the Cu surface, increasing active carbon on Cu surface can directly enhance the graphene growth rate. However, the high carbon species concentration on the Cu surface also leads to more nucleation and thus smaller graphene domain sizes. People desire large graphene domains and try to have little active carbons on Cu surface to suppress the nucleation density with the compromise of growth rate. That is the reason why the reported growth rate is usually very low on Cu. It seems to be a dilemma in optimizing both the domain size and growth rate. A good way to get out of this dilemma is to use Cu‐Ni alloy substrate. Ni has higher catalytic activity and carbon solubility than Cu and can be alloyed with Cu with any element content, thus changing the growth behaviors.The metallic substrate in the CVD graphene growth also serves as catalyst for the CH4 would have high colliding frequency and substantially enhance the local concentration of carbon flux, and the hence graphene growth rate is improved.Besides the composition, the configuration of substrate also affects the growth rate distinctly. When rolling or vertically stacking Cu foils via direct physical contact with a gap of about tens of micrometres, the gas flow in the narrow spaces of the stacked Cu foils could be in the molecular flow regime, where the CHAlso the surface properties, including surface index, step, bunches, impurities and defects, have dramatic influence on the graphene growth. Generally, Cu foils used to grow graphene are polycrystalline and on different facet surfaces graphene growth behaviours are different. The growth rate on Cu(111) is higher than (100) and other high index facets, attributed to higher diffusion and improved absorption of hydrocarbon precursor.3.24 is typically used as the carbon source for graphene growth and thus can affect the growth rate directly. Apparently a higher concentration of CH4 would lead to faster growth of graphene over the Cu surface, while it also results in higher nucleation density. The nucleation occurs when the C species on the active sites are oversaturated and if the CH4 flow is too low that there will be no nucleation and growth.4 feeding, namely low feeding at the nucleation stage and high feeding in the growth stage, to achieve lower nucleation density but large graphene domain size.CH2 also plays vital roles in the graphene growth process. Actually, Cu can catalyse hydrogen dissociation reaction as well.2 would reduce surface sites, leaving fewer available Cu sites for CHx (x = 1–4) dissociative chemisorption. In addition, the dissociated H atoms are active to etch the edge carbon atom of existing graphene domains.2 is impeditive to the rapid growth, it is essential to achieve monolayer growth.HFigureAs discussed above, growth process is a balance between carbon diffusion/deposition and hydrogen etching , is thermodynamically unfavourable and the reaction barrier is relative high.x–1) may combine with each other and finally form H‐terminated graphene domains. Thus further C species edge attachment and lattice incorporation for the graphene domain growing up require dehydrogenation. In a typical graphene growth process, the growth rate can be either diffusion‐limited (insufficient active C supply) or edge‐attachment‐limited.In the growth process, the dissociation of CH4 via the reaction CHx + O → Cu  CHx–1 + OH . As shown in Figure4 decomposition, the O atom on the Cu surface is able to interact with the H atom by the formation of O–H bond3 radicals by several orders of magnitude. Considering that a CH4 molecule decomposition is a complicated process, we carefully evaluated the full dehydrogenation of CH4 by Climbing Image‐Nudged Elastic Band (CI‐NEB) calculation and the result is presented in Figure 3, CH2, CH and C, on the metal surface and/or in the gas phase. Thus, no matter which kind of C species should be the key participant of the graphene growth, a quick supply of C source around each graphene domain is guaranteed.It was reported the O on Cu surface can reduce the energy barrier to the dissociation of CHAlso, given that the H on the Cu has lower energy in the form of OH compared with that of H on Cu3.44 is chemically stable. Therefore, the temperature must be much higher using Cu as substrate and CH4 as carbon source. Usually, 1000 °C or above is applied to achieve the growth of high‐quality single‐crystal graphene. At lower temperature, like 800 °C, graphene can be synthesized but with poor quality.4, diffusion of active carbon and attachment of C species onto graphene nuclei. Also, higher temperature can decrease the nucleation density of graphene.As an active factor in the CVD growth of graphene, temperature can affect the growth rate significantly. The catalytic activity of Cu is lower compared to other mentioned metal catalyst and CH4 on Cu surface is enhanced.4.4 flux is high, the self‐limited growth process would be broken and multilayer graphene may form.4 is not a good choice for the growing of high‐quality graphene.Pressure is another important factor in the growth process. It has been widely accepted that low pressure CVD can readily achieve uniform monolayer graphene film. At lower pressure, the lower gas flux results in fewer collisions and a higher diffusivity coefficient, and thus the absorption of CHBased on the above discussion, the effects of temperature and pressure on growth rate are interactive. Appropriate temperature and pressure conditions can be reached to produce graphene with high growth rate and good quality.4To improve the growth rate of CVD graphene is essential for both fundamental research and industrial applications. In this review, the available ways towards the ultrafast graphene growth are discussed in details. Proper catalytic substrate can achieve the higher growth rate, while it is more powerful in the control of layer number. The optimization of feeding gas ratio has limited capacity to improve the grow rate. The deduction of reaction barrier, for example by the introduction of oxygen, can improve the growth rate exponentially and turns out to be a very powerful way to realize ultrafast growth.In the future, further work is required for faster growth and larger graphene single crystals. Several possible routes are proposed: (i) to explore a more efficient way to reduce the reaction barrier for the graphene growth; (ii) to epitaxially grow well aligned graphene domains and make them seamlessly stitch together into a complete single‐crystal film; (iii) to combine with roll‐to‐roll technique to realize streamline production. Route (i) can be realized by effective introduction of oxidative substance like O onto Cu surface, to enhance the dissociation of hydrocarbon. This is expected to readily obtain millimetre‐scale single‐crystal graphene islands in several minutes. Actually, route (ii) has been implemented by growing graphene on Ge(110)The authors declare no conflict of interest.


In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17–36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV.  Papillomaviruses (PV) are a diverse group of non-enveloped, double-stranded, species-specific DNA viruses with strict epithelial tissue tropism. More than 200 human PV (HPV) genotypes have been completely sequenced and classified into five genera: alpha, beta, gamma, mu and nu  . While gA group of HPV types belonging to genus beta are hypothesized to play a role as adjunct to the main carcinogen UV light in the development of non-melanoma skin cancers (NMSC) in immunosuppressed OTR and HIV-positive patients. Although beta HPV are detected in skin swabs or hair bulbs from normal individuals by highly sensitive nested PCR, infections appear to be contained by the immune system. Beta HPV have originally been identified in patients with the rare genodermatosis Epidermodysplasia verruciformis (EV), who typically present with generalized flat warts and scaly plaques. Early in life EV patients develop multiple NMSC, squamous cell carcinomas (SCC) or basal cell carcinomas (BCC), predominantly on sun-exposed areas . The genWhile the role of beta HPV causing NMSC in EV patients is long established, their role in development of NMSC in OTR is less clear. Beta HPV infections of the skin are acquired very early in life, even a few days after birth, reaching an overall prevalence of approximately 90% with increasing age without causing clinical symptoms. Studies have revealed increased beta HPV DNA carriage in cutaneous SCC compared to perilesional skin, whereas prevalence and viral load appear higher in precancerous actinic keratoses, opposite to the findings in EV patients –14. In c®), HPV16/18/6/11 , or HPV16/18/31/33/45/52/58/6/11 , with the potential to protect against 70–90% of all CxCa cases and, for the latter two vaccines against 90% of genital warts. However, they do not protect against the mucosal types associated with the remaining 10% of CxCa cases requiring cervical screening programs to be maintained in vaccinated women  and CsCl-PBS [29% (wt/wt)] density gradients .Viral fusion proteins were identified by SDS-PAGE / Coomassie staining or Western Blot using mAb Camvir-1   or a HPV2 +0.02% Tween 80. Fifty μg of respective chimeric VLP were adjuvanted with 500μg alum plus 50μg MPL  and used to immunize two NZW   each in a 5-dose regimen at week 0-3/4-6-8-16 for HPV1L1-4RG1 VLP, or in a 4-dose regime at week 0-4-6-8 for HPV16L1-17RG1, HPV5L1-17RG1, 16L1-5L2(aa53-72) or 18L1-5L2(aa53-72) VLP, and sera drawn two weeks after the final boost.Gradient-purified proteins were extensively dialyzed against PBS +0,5M NaCl +1mM CaCl3 (pH 10.6) +0.01M freshly added dithiothreitol (DTT) buffer over night at 37°C. The next day wells were washed, blocked with 0.5% milk/PBS, and serial dilutions of antibodies  added in triplicates for one hour at room temperature (RT). Antibodies used were neutralizing anti HPV16 L1 mouse mAb H16.V5, H16.5A, H16.14J, 263.A2, H16.E70, or neutralizing anti HPV18 L1 mAb H18.J4 , or non-neutralizing mAb Camvir-1 directed to a linear HPV16 L1 epitope (BD Pharmingen), or rabbit serum raised against HPV16 L2 aa11-200 . Chimeric HPV5L1-17RG1 and HPV1L1-4RG1 VLP were evaluated using the HPV16L2 aa11-200 polyclonal serum as no Ab to HPV1 or HPV5 L1 were available. After a PBS wash step, the second antibody goat anti-mouse or goat anti-rabbit IgG-HRP (Bio-Rad) was added at 1:10,000 dilution for 45 minutes at RT, followed by substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (Roche). The OD at 405nm was determined with an ELISA reader  and positive titers are reported for OD values greater than the mean OD of pre-immune sera plus 3 standard deviation (SD).Antigenicity of chimeric L1-RG1 or L1-L2(aa53-72) VLP as compared to wt HPV16L1 or wt HPV18L1/L2 VLP was analyzed by ELISA. VLP preparations were quantified by SDS-PAGE Coomassie staining by visually comparing the intensity of the L1 band at ~50 kDa to serial 1:2 dilutions (from 1 mg/ml to 0.125 mg/ml) of a bovine serum albumin standard (possible impurities or degraded L1 proteins were not taken into account). In short, 0.1μg native VLP per well in 100μl PBS were attached onto Maxisorp 96-well plates (Nunc) over night at 4°C. For denaturing conditions, 0.1μg VLP were dried onto the ELISA plate without a lid in 0.2M NaHCOTo evaluate immunogenic display of the genetically inserted L2 epitopes on the VLPs’ surface, peptide ELISAs were performed using biotinylated RG1-homology peptides of either HPV17 or HPV4, or the HPV5 L2 epitope aa53-72 . Briefly, 1μg biotinylated peptide in 100μl coating buffer (1M Tris/HCl (pH7.4) +1M NaCl +0.001% Tween-20) per well were plated at 4°C overnight onto Streptavidin plates (Nunc immobilizer Streptavidin F96 clear). Wells were then washed with coating buffer for 5 minutes and blocked with 1% milk/PBS at 4°C overnight, washed again and incubated in triplicates with serial dilutions of antisera  for one hour at RT on an ELISA plate shaker. Pre-immune or immune sera raised against wt HPV18L1, HPV16L1, HPV5L1 VLP, or a human HPV1-reactive serum were used as controls. The second antibody goat anti-rabbit IgG-HRP (Bio-Rad) was added for 45 minutes at dilution 1:10,000 at RT, prior to adding the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (Roche). The OD at 405nm was determined as stated above and titers are reported for values greater than the mean OD of pre-immune sera plus 3 standard deviation (SD).https://home.ccr.cancer.gov/Lco/codonmodification.htm), further edited by hand, synthesized  and sub-cloned into the double expression vector pVitro1-neo-mcs (Invivogen). The BamHI-XbaI flanked L1 was cloned into MCS2 using the BglII-NheI sites, while the BamHI-XbaI flanked L2 gene was inserted into MCS1 using the BamHI-AvrII sites , HPV17 , HPV20 (U31778), HPV23 (U31781), HPV24 (U31782), HPV36 (U31785), HPV37 (U31786), HPV49 (X74480), HPV80 (Y15176), HPV92 AF531420), HPV93 (AY382778) and HPV96 (AY382779) were designed according to published instructions . L1 and 1420, HPV6 293TT cells were plated into 175cm2 flasks and one day later transfected with 38μg DNA each of HPV L1 and L2 expression and reporter plasmids . On the next day media was changed, and 48 hours after transfection cells were lysed in PBS +9.5mM MgCl2 +0.5% volume Triton-X100 +0.25% Ammonium sulfate +0,1% Benzonase (of a 25U/μl stock) +0,1% Plasmid Safe. For particle maturation cell lysates were incubated 20–24 hours at 37°C. Finally, PsV were salt extracted and purified by ultracentrifugation on Optiprep step gradients .PsV were produced in 293TT cells as described , 55. BriPlasmids for L1 and L2 of HPV16 and HPV18 were provided by J. Schiller, NCI; for HPV5 and 45 by C. Buck, NCI; for HPV3 and 76 by J. Dillner, Karolinska Institute; for HPV27 and 57 from ; and forExpression of L1 and L2 proteins of novel beta PsV was verified by SDS-Coomassie staining and Western Blot using either mAb Camvir-1 (BD Pharmingen) , mAb AU1In order to generate type-specific control immune sera, one NZW rabbit each was immunized with HPV4, 20, 24, 36, 38, 39, 59, 68, 76 and 96 in a 3-dose regime at week 0-3-6 using 20μg PsV and Freund’s adjuvants (CFA/IFA) and sera finally drawn two weeks after the final boost (Charles River Laboratories). Additionally, HPV15, 23, 37, 49 and 80 PsV were used to immunize groups of mice (n = 5) subcutaneously with 50ng PsV plus alum  as adjuvant in a 2-dose regime (week 0–2) and sera were drawn and pooled two week after the final boost.®, Life Technologies), subcloned into pET28a vector and, and used to transform Rosetta DE3 cells (Novagen). Protein expression was induced by IPTG  as per company’s instructions and purified via its His-tag using NTA columns . 300μg peptide adjuvanted with CFA/IFA were used to immunize two NZW in a 3-dose regime at week 0-3-6, and final immune sera drawn two weeks after the final immunization.A gene encoding 17RG1x5 peptide  was synthesized (GeneArt4 293TT cells per well were plated onto a 96-well plate and infected with SEAP-containing PsV that were pre-incubated without or with (pre-) immune sera on ice, for one hour. Infection was assessed three days later by measuring the SEAP signal at 405nm  after addition of 4-nitrophenyl phosphate disodium salt hexahydrate  dissolved in diethanolamine (Sigma). Additionally, neutralization assays of types that showed low infectivity  were evaluated using Ziva Ultra SEAP Plus Kit (Jaden BioScience) or GreatEscape SEAP chemiluminescence kit  according to company’s instructions. In short, after 72 hour incubation, plates were shaken for a minute to gain a homogenous supernatant prior centrifugation for 5 minutes at 1700xg. 5 or 20μl were then transferred onto an opaque 96-well Optiplate (Nunc) containing 75μl 1x SEAP Sample Preparation Solution. Plates were shaken again and incubated at 65–68°C for 50 minutes. Following this inactivation of endogenous SEAP, 25μl (for the ZIVA Ultra SEAP Plus Kit) or 100μl (for the GreatEscape SEAP Kit) of substrate were added and plates incubated in the dark for 30 minutes prior reading luminescence for 0.2–0.3 seconds per well using either the Victor3 or Fluoroskan Ascent FL plate reader. Serum dilutions showing at least a 50% reduction in SEAP signal compared to pre-serum were considered to be neutralizing.The L1-based assay was performed as previously described . Briefly6 HaCaT cells were plated onto 96-well plates for 24 hours at 37°C, cells were washed twice with PBS and lysed using PBS +0.5% Triton X-100 +20mM NH4OH for 5 minutes at 37°C. Following lysis, 100μl PBS were added and removed again for three times to gently wash the remaining extracellular matrix, and PsV in 120μl/well CHOΔfurin conditioned medium containing 5μg/ml heparin (Gilvasan Pharma) added. The ECM/PsV mix was incubated overnight at 37°C and the next day washed with PBS to remove non-attached PsV. Antibody serial dilutions were added and incubated at 37°C for at least 1 hour. After this incubation step, 8*103 PGSA-745 cells were directly added to the wells (50μl/well) and incubated at 37° for two days. For luciferase encoding PsV, assays were evaluated using the Luciferase Assay System (Promega #E1501) and the Cell Culture Lysis Reagent (Promega #E153A) according company’s instructions in 96-well opaque Optiplates (Nunc). Luciferase activity was measured using either the 1420 Victor3 Multilabel Counter (PerkinElmer) or the Fluoroskan Ascent FL (Thermo Scientific) plate reader, with 10 second per well reading time. For SEAP encoding PsV, assays were evaluated using the Ziva Ultra SEAP Plus Kit (Jaden BioScience) or GreatEscAPe™ SEAP Kit  as mentioned before. For CHOΔfurin conditioned medium, 1*106 cells were grown in 17ml media and supernatants harvested after incubation at 37°C for 4 days. Serum dilutions showing at least a 50% reduction in SEAP signal compared to pre-serum were considered to be neutralizing. PGSA-745 and CHOΔfurin cells were kindly provided by Patricia Day, NCI.The L2-based assay was performed according to Day et al . In shormg/ml; Promega; 10 minute exposure) and imaging by the IVIS system . Data were analyzed by the Living image software program by drawing uniform regions of interest (ROI) around the luminescence emitting genital area of each mouse to determine the average radiance within the ROI. A group of mice (n = 5) was challenge with CMC only to determine background radiance. Luciferase activity was measured as p/s/cm2/sr (average radiance).Six to eight weeks-old female Balb/c were obtained from Charles River Laboratory and the challenge experiments were performed according to Roberts et al. . BrieflyThe cutaneous challenge was performed according Wu et al. . BrieflyAnimal studies were approved by the ethics committee of the Medical University of Vienna and the Austrian Federal Ministry of Science and Research (BMWF-66.009/0173-1l/3b/2011) and animal care was in accordance to the guidelines of the Institute for Biomedical Research Vienna. The vaginal/cutaneous challenges were performed under Ketaminhydrochlorid/Xylazin anesthesia to minimize suffering. Animals were sacrificed by cervical dislocation.In vivo statistical analysis was performed using Graph Pad Prism to evaluate p-values between indicated groups .Chimeric L1-L2 fusion genes were designed that encode L1 capsid proteins of hr mucosal HPV16 or HPV18, hr cutaneous beta HPV5, or common cutaneous HPV1 L1 as scaffold to present L2 peptides via the DE surface loop. The HPV17 (17RG1) and HPV4 (4RG1) L2 peptides, comprising each N-terminal 20 aa14-33, were chosen based on homology to the highly conserved broad cross-neutralization epitope ‘RG1’ of HPV16 L2 . The HPVRecombinant baculoviruses were isolated by plaque purification, amplified, and fusion proteins expressed by infection of Sf9 cells and verified by SDS-PAGE. By Coomassie staining, the five fusion proteins migrated around 50kDa . AdditioLarge molecular weight complexes were purified from infected Sf-9 cell extracts on density gradients, negatively stained by uranyl acetate and analysed by transmission electron microscopy (TEM). Electron micrographs showed spherical particles around 50-60nm for chimeric HPV5L1-17RG1 (A), HPV16L1-17RG1 (B), HPV1L1-4RG1 (C), HPV16L1-5L2(aa53-72) (D) and HPV18L1-5L2(aa53-72) (E) fusion proteins , indicatChimeric VLP were further analysed by ELISA for antigenicity of the VLP scaffold and ability to present inserted L2 epitopes. Under native conditions, HPV16-neutralizing mAb H16.E70 did not bind and H16.V5 only weakly bound to chimeric HPV16L1-17RG1VLP, whereas both mAbs reacted with wt HPV16 L1 VLP as expected  left, whNext, HPV16L1-5L2(aa53-72) VLP were examined by ELISA and compared to wt HPV16 L1+L2 VLP using conformation-dependent HPV16-neutralizing mAb. Similar to above results, neutralizing mAb binding to native chimeric VLP was weak as compared to wt HPV16 L1+L2 VLP, indicating that epitope insertion interfered with mAb binding . As expeFor chimeric HPV1L1-4RG1 VLP mAb Camvir-1 showed a similar binding pattern by ELISA as seen for the other chimeric VLP, with stronger reactivity to denatured than native VLP , whereasTo generate the tools for evaluating the potential of chimeric VLP immunogens to protect against beta HPV infections, expression vectors for 12 beta HPV PsV types were generated using respective codon-optimized L1 and L2 genes. These comprised HPV15, 17, 20, 23, 24, 36, 37, 49, 80, 92, 93 and 96, representing beta HPV species 1–5 and included types identified in skin cancer samples. Following transfection into 293TT cells and PsV purification on density gradients, L1 plus L2 capsid proteins were verified by Western Blot using mAb to L1, or polyclonal sera raised against heterologous beta PsV types if mAb were non-reactive. MAb Camvir-1 recognized HPV92 and HPV17 L1 proteins (and HPV16 L1 VLP used as positive control) , whereasFurther, assembly of beta HPV L1+L2 proteins into capsids was assessed by TEM of negatively stained PsV preparations. While spherical structures were observed for HPV15, 17, 20, 23, 24, 36, 37, 49, 80, 92 and 96, no capsids were visible for HPV93 PsV preparations . Of notein vitro neutralization assays, and those with high in vitro infectivity were tested in vivo in vaginal mouse challenges. HPV17 and 93 proved to be non-infectious in vitro even though HPV17 (but not HPV93) preparations showed numerous and correctly sized virions by TEM  and HPV18L1-5L2(aa53-72), high neutralization titers were detected against homologous L1-scaffold HPV16  and HPV18  by L1-PBNA , no  were detected by L2-PBNA against HPV4 only (a PBNA for HPV1 is not available), but not against any of the tested common cutaneous  or beta HPV types tested  by L1-PBNA .in vitro analysis for cross-neutralization, one antiserum each to HPV5L1-17RG1 (serum #2), HPV16L1-17RG1 (serum #3) and HPV1L1-4RG1 VLP (serum #2) was further evaluated for in vivo cross-protection using the murine vaginal challenge model [Following ge model .Passive transfer of HPV5L1-17RG1 VLP-raised serum protected mice partially from experimental challenge with HPV20, 24 and 96  against any of the tested cutaneous HPV except HPV4, and thus in vivo challenge was performed with this type only. As can be seen in For sera induced by HPV1L1-4RG1 VLP vaccination, While the involvement of beta HPV in NMSC carcinogenesis in immunosuppressed patients is supported by epidemiological and experimental data, a possible role in keratinocyte skin cancer of the general population is less clear. Beta HPV DNA is more frequently detected in premalignant actinic keratosis than in NMSC and only a minority of cancer cells harbor viral DNA, supporting a possible role in initiation but not maintenance of the tumor phenotype .In a recent study using the natural Mastomys natalensis papillomavirus (MnPV) mouse model, prophylactic vaccination of naïve mice, and even therapeutic vaccination of previously infected animals protected against development of MnPV-associated skin cancer . TherapeWe have used the RG1 homologs of beta HPV or a common cutaneous type as type-common experimental vaccine antigens to more directly target cutaneous HPV, compared to a previous HPV16-based chimeric RG1-VLP (HPV16L1-16RG1) immunogen , 47. ThePreviously, expression of recombinant HPV L1 proteins in insect cells has shown that truncation of the nuclear targeting and DNA binding signals at the C-terminus of HPV16 and HPV18 L1 reduced incorporation of cellular DNA, whereas the capacity to assemble in immunogenic VLP was retained . In addiThe current standard approach to assess neutralizing antibodies, which are the main effectors of protection induced by L1 VLP vaccination, is by ‘L1- sensitive’ PsV-based neutralization assays (L1-PBNA). However, the L1-PBNA was shown to be less sensitive and thereby possibly underestimating the extent of L2-mediated cross-protective humoral responses. Consequently, a novel L2-based neutralization assay that detects L2-mediated neutralization (L2-PBNA) with higher sensitivity, as well as L1-targeted neutralization, was employed to analyse the (cross-) neutralization spectrum of chimeric VLP .in vitro which were not protective against HPV5 PsV challenge in vivo, while cross-neutralization was observed against additional 5 of 13 beta HPV by L2-PBNA. Nevertheless, mice passively immunized with sera raised against chimeric HPV5L1-17RG1 VLP were partially protected against experimental challenge with HPV20, 24 and 96 PsV, but not against HPV38 and 76. These results suggest that 17RG1 epitope insertion into and sequence replacement of the DE loop disrupted or sterically hindered major HPV5 L1 neutralization epitope(s) exposure in the chimeric HPV5L1-17RG1 VLP. Unfortunately, mAb to analyze chimeric and parental HPV5 VLP for type-specific neutralization epitopes were not available. However, the binding of HPV16-neutralizing mAb H16.V5 was recently mapped to 17 residues found in five intertwined loops of two neighboring L1 molecules, comprising the DE and HI loops of one L1 and the BC, DE and FG loops of an adjacent L1 [Unexpectedly, for antisera to HPV5L1-17RG1 VLP low type-specific neutralizing titers to homologous HPV5 were detected acent L1 , 67. Invacent L1 .in vivo protection against HPV16 PsV challenge, indicating the chimeric HPV16 VLP retained protective immunogenicity. RG1 epitope insertion at L1 positions 136/137 might have disrupted the DE loop involvement in the H16.V5 recognition site, while residual binding might be due to the remaining FG residues. MAb 263.A2 appears to have a similar binding pattern to that of H16.V5, with FG and HI loop residues recognized by both mAb defining a complex epitope recognized by a majority of human sera in response to natural HPV16 infection [In vivo experiments further confirmed immunogen-mediated (cross-) protection against challenge with HPV20, 96 and the homologous HPV16, while partial protection was observed against HPV5, 24 and 38.Neutralizing mAb H16.V5 has been shown to block 75% of reactive human sera with HPV16 VLP. Although reactivity of H16.V5 with HPV16L1-17RG1 VLP was rather weak, immune sera conferred complete type-specific nfection , 68. No nfection , 70. Of in vivo protection by murine vaginal challenge, because infection at the cutaneous site with beta HPV5, a type showing usually high in vivo infectivity, was much lower and more variable when compared to infection of the vaginal mucosal and thus did not allow robust analysis of vaccine efficacy  revealed that 12 C-terminal aa of its L2 protein were predicted to be missing (residues 525–536: SLKKRKRKRKYL) by comparison to the HPV17 L2 sequence entry from the HPV PAVE homepage . The missing sequence is highly conserved to the C-terminus of HPV16 L2 that contains a nuclear localization signal (aa454-462), a nuclear export signal (aa462-471) and a dynein-binding site  [Novel beta HPV PsV types were designed according to HPV prevalence in NMSC of both immune-suppressed and immune—competent patients, and to provide at least one type representative of different beta HPV species . In genel 40 aa) . MutatioThe second L2 epitope used in this study is the HPV5 L2 aa53-72 homolog of HPV16 L2 aa56-75. The latter epitope has originally been described to induce cross-neutralizing antibodies against mucosal hr types . More rein vitro and partially protected in vivo against HPV4 challenge, likely by targeting the homologous HPV4 RG1 epitope. However, antisera failed to cross-neutralize distantly related cutaneous alpha and beta HPV by PBNA, which is likely due to ≤50% RG1 sequence homology to tested cutaneous types . Antisera were neutralizing us types .In conclusion, chimeric VLP immunogens that display cross-neutralization L2-RG1 epitopes of cutaneous HPV types are evolving as a promising tool to develop next generation L2-based prophylactic vaccines with broad-spectrum protection against HPV types associated with skin pathologies in immunocompetent and immunosuppressed populations.S1 Fig(A) Reactivity of non-neutralizing mAb Camvir-1 and HPV16-neutralizing mAb H16.5A, H16.14J, 263.A2 with native or denatured HPV16L1-17RG1 VLP. Analysis of L2 epitope presentation by HPV16L1-17RG1 VLP (B) and HPV5L1-17RG1 VLP (C) using Camvir-1 and an antiserum raised to HPV16 L2 aa11-200 under native and denatured conditions.(TIF)Click here for additional data file.S2 Fig(A) HPV16L1-5L2(aa53-72) and (B) HPV18L1-5L2(aa53-72) VLP were compared to wt L1+L2 VLP under denatured conditions using indicated mAb or polyclonal serum. (C) Native or denature HPV1L1-4RG1 VLP were analyzed with Camvir-1 and antiserum to HPV16 L2aa11-200.(TIF)Click here for additional data file.S3 FigGroups of Balb/c (n = 10) were passively immunized with pre-immune serum and infected with 40μl HPV5 PsV at the skin of the belly or the vaginal mucosa as described in Materials. After subcutaneous or intravaginal addition of D-luciferin, bioluminescence was evaluated by in vivo imaging. Data from the vaginal challenge was taken from (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 TableMAb Camvir-1 or AU1, or polyclonal sera raised against the indicated L1 and/or L2 antigens, were used to detect L1 and L2 expression of a panel of beta HPV PsV by Western blot. Results are summarized. (-), (~) and (+) indicate negative, weak, or positive immunoreactivity, respectively. Empty boxes indicate that no experiment was done.(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S5 Table(TIF)Click here for additional data file.


In this paper, the periodic double-layer graphene ribbon arrays placed near a metallic ground plate coated by a dielectric layer are proposed and analyzed by the coupled-mode theory (CMT) to predict the perfect absorption response in the mid-infrared region. Numerical simulations of the finite-difference time-domain (FDTD) method confirm this effect and give the underlying physical origin. The anti-symmetric dipole-dipole coupling mode is supported by the double-layer graphene ribbons and acts as the electrical resonance to suppress the reflection, because of the impedance matching. The transmission from this system is restricted by the ultra-thick metallic ground plate. All incident electromagnetic energy is efficiently confined in the interlayer between graphene ribbons and the metallic plate, and the dramatic narrowband perfect absorption peak with the FWHM  of 300 nm hence is achieved. The spectral position of the absorption peak can be dynamically tuned by a small change in the chemical potential of graphene, in addition to varying geometrical parameters of the absorber. Meanwhile, this device exhibits good absorption stability over a wide angle range of incidence around ± 60° at least. Such absorber will benefit the fabrication of mid-infrared nano-photonic devices for optical filtering and storage. Over the past decades, an extensive amount of research has been carried out on the metamaterials19111518192022Recently, graphene24272830313234Currently, a great diversity of graphene-based metamaterials3738x-y cross-section of this device with the corresponding geometrical description is shown in the inset. The double-layer graphene ribbons with identical width of W are separated by the silica layer with the thickness of d. The period of the ribbons array is P. The cavity between the metallic ground plate and the lower graphene layer is filled also by the silica layer with thickness of D. The thickness of the metallic ground plate is H. When the plane wave with the polarization along the x axis irradiates normally this structure, the second-order dipole resonance should form on the single graphene ribbond = 50 nm, the anti-symmetric plasmonic dipole-dipole coupling resonanceT = 0), as the thickness of the metallic ground plate with H = 800 nm is much larger than the penetration depth of electromagnetic waves. When the double-layer graphene ribbons are considered as a whole and called the anti-symmetric dipole-dipole coupling resonator, the proposed device behaves as such construction of the single port coupled with a resonator. The versatile CMT4647First, the structure of the proposed absorber composed of the periodic double-layer graphene ribbon array placed near a metallic ground plate coated by a dielectric layer is depicted schematically in the S+, and S−, respectively. The resonant frequency of the anti-symmetric dipole-dipole coupling mode formed between double-layer graphene ribbons is ω0. The dissipative and radiative losses occurring in the anti-symmetric coupling resonator are represented by τa−1 and τr−1, respectively. The coupling strength between the incident wave and the anti-symmetric resonator is characterized by τc−1. When the incident wave with the frequency ω is launched into this system, the time-evolution-normalized amplitude of the anti-symmetric dipole-dipole coupling resonator is expressed asAs shown in the inset of Thanks to the power conservation and time reversal symmetry, the relationship between the amplitudes of the normalized incoming and outgoing waves transmitted through the only port is described asr0 is the reflectance of this system without the double-layer graphene ribbon array. Because the metallic ground plate behaves as a mirror, all incident waves will be reflected directly at the upper surface of the metallic plate. Therefore, we can obtain r0 = −1. j stands for the imaginary unit. Isolating the S−/S+ and going to the frequency domain ejωt+ , the reflectance of the device can be obtained and expressed asHere, the ω = ω0 for the reflection spectrum. At the ω = ω0, the effective impedance of this system is z0 = 1), the radiative losses can be neglected, corresponding to τr−1 ≈ 0. Hence, only the condition of τa−1 = τc−1 should be satisfied. At the ω = ω0, the impedance matching is realized and r = 0. Thus, the absorption is A = 1 − T − |r|2 = 1. In other word, the perfect absorption response will be achieved at the ω = ω0.The impedance of this system can be defined as The reflection of this system isThe absorption of this structure can be given byτa−1 = τc−1 = 1*1012 Hz are considered for obtaining a useful impedance matching. The resonant wavelength λ0 = 2πc/ω0 is assumed as 11.16 μm, here c is the velocity of light in vacuum. Obviously, the perfect absorption feature occurs. In order to confirm the CMT-predicted perfect absorption response and further investigate comprehensively the proposed absorber, the numerical simulation is performed by the Lumerical FDTD Solutions.The CMT predicted absorption spectrum is shown in y direction). The surface conductivity (σg) of the graphene is governed by the Kubo formula49τ, temperature T, chemical potential μc, and incident wavelength λ (frequency ω). At the room temperature and for the highly doped graphene in the simulated mid-infrared spectral range, the chemical potential is always above half of the photon energy. The Kubo equation hence is reduced toεeq of graphene is thickness-dependentμc = 0.4 eV, and the momentum relaxation time is chosen as τ = 0.5 ps. The metal is chosen as the silver with the dispersive permittivity characterized by the Drude model5354ε∞ = 3.7 is the dielectric constant at infinite angular frequency; ωp = 9.1 eV is the bulk plasma frequency representing the natural frequency of the oscillations of free conduction electrons; γ = 0.018 eV stands for the damping frequency of the oscillations. In the considered mid-infrared region, the silica is assumed to be a non-dispersive dielectric with the index of 1.45. The unit cell with P = 300 nm, W = 150 nm, and D = 600 nm is performed. Periodic boundary conditions are applied along the y directions and perfectly matched layers are employed in the x direction. A plane wave with the electric field parallel to the x axis illuminates normally the periodic structure. We use the non-uniform mesh, and the minimum mesh size inside the graphene layer equals 0.1 nm and gradually increases outside the graphene sheet, for saving storage space and computing time. 2D-FDTD simulated results are presented in In the numerical calculations, the graphene ribbon is modeled as an ultra-thin film with a thickness of Δ  distributions of the entire unit cell at wavelengths of λ0 = 11.16 μm and λ = 14.00 μm, are shown in λ0 = 11.16 μm, the electrical resonance yields at the anti-symmetric coupling plasmonic resonator formed by the double-layer graphene ribbons. The effective impedance of the vertically coupling graphene ribbons matches to that of the free space, and the reflection thus is suppressed. The transmission from this system is also inhibited due to the ultra-thick metallic ground plate. All incident electromagnetic energy is efficiently confined in the silica interlayer between the graphene ribbons and the metallic ground plate. Therefore, the perfect absorption feature is achieved by the simple graphene-based structure. On the contrary, the electrical resonance of the double-layer graphene ribbons and the effective impedance matching condition cannot be formed at the other nor-resonant wavelengths. The most incident power is reflected and cannot be confined by this device, as shown in λ = 14.00 μm.In the d = 50 nm and the duty ratio of W/P = 0.5 are fixed. Other structural and material parameters are unchanged. The structure with the simultaneous change in the width W of double-layer ribbons is simulated and results are presented in W = 150, 180, 210, and 240 nm are offered. The obvious width-dependent tunability is achieved. In addition, the real part of the wave vector of the anti-symmetric dipole-dipole coupling resonance increases with the vertical coupling distance between ribbons increased increases. Simulated absorption spectra with different coupling distances are displayed in Now that the spectral position of the perfect absorption peak corresponds to the resonant wavelength of the anti-symmetric coupling dipole-dipole resonator formed on the double-layer ribbons, the change in the resonant wavelength of the anti-symmetric coupling resonator can be utilized to tune the spectral position of the absorption peak. For anti-symmetric coupling dipole-dipole resonator, the changes in the geometrical parameters of double-layer graphene ribbons should have a direct effect on its resonant wavelength. Thus, the changes in the geometrical parameters of graphene ribbons can be used first for tune the spectral position of the absorption peak D are presented in D has little effect on the central wavelength of the absorption peak, whereas influences greatly the absorption and FWHM of the resonant peak. In other word, the absorption spectrum can be modulated by changing the thickness D, without changing the spectral position of the absorption peak. This phenomenon offered by the D of the silica spaceD increases from 50 to 600 nm, the perfect impedance matching to that of the free space is formed gradually. With the thickness D increase further, the perfect impedance matching is broken. Hence, the perfect absorber is achieved first and then is hampered with the increase of the thickness D. On the other hand, the anti-symmetric dipole-dipole coupling plasmonic resonance in double-layer graphene ribbons is localized around the graphene ribbons with deep sub-wavelength nano-scales, which are less than 50 nm as shown in the inset of D increases from 50 to 1000 nm.In addition, for the absorption peak, the most incident electromagnetic power is confined in the space between the lower graphene ribbon and the metallic ground plate. Hence, the height of the space should have a direct effect on the performance of this absorber. Numerical absorption spectra under different distances μc = 0.35, 0.4, 0.45, and 0.5 eV, respectively, are shown in Besides the geometrical tunability, the graphene-based absorber is particular to own the novel gate-voltage-dependent feature that the chemical potential of graphene can be dynamically tuned by applying external gate voltages, as shown in It should be pointed out that the electromagnetic waves irradiate usually this absorber with an oblique incident angle in the practical applications. Thus, it is necessary to investigate the robustness of the proposed perfect absorber under the oblique incidence. Other simulation parameters are similar to that in To sum up, the periodic double-layer graphene ribbon arrays placed on a metallic ground plate coated by a dielectric space are proposed and investigated both numerically and theoretically. The well-known CMT is used for analyzing the structural property and predicting the perfect absorption feature. FDTD numerical simulation is utilized to give the underlying physics. Simulated results exhibit that the localized anti-symmetric dipole-dipole coupling modes occurs at double-layer graphene ribbons and acts as the electrical resonance to suppress the reflection, thanks to the impedance matching. The ultra-thick metallic ground plate is used for restricting the transmission from this system. The incident electromagnetic energy is efficiently confined in the interlayer between graphene ribbons and the metallic substrate, and the novel perfect absorber hence is achieved. The spectral position of the absorption peak can be dynamically tuned by a small change in the chemical potential, in addition to varying geometrical parameters of the absorber. Meanwhile, this device exhibits good absorption stability over a wide angle range of incidence around ± 60° at least. Such simple plasmonic perfect absorber may benefit the fabrication of nanophotonic devices for optical filtering and storage in the mid-infrared region.y directions and perfectly matched layers are employed in the x direction. A plane wave with the electric field parallel to the x axis illuminates normally the periodic structure. The non-uniform mesh is adopted, and the minimum mesh size inside the graphene layer equals 0.1 nm and gradually increases outside the graphene sheet, for saving storage space and computing time.The 2D finite-difference time-domain (FDTD) method is used for the numerical simulation and the commercial software of Lumerical FDTD Solutions is performed. Periodic boundary conditions are applied along the How to cite this article: Li, H. et al. Tunable graphene-based mid-infrared plasmonic wide-angle narrowband perfect absorber. Sci. Rep.6, 36651; doi: 10.1038/srep36651 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Testicular germ cell tumors (TGCTs) are nearly universally curable malignancies. Nevertheless, standard cisplatin-based chemotherapy is not curative in a small subgroup of patients. Previously, we showed that PD-L1 overexpression is associated with worse prognosis in TGCTs, while tumor infiltrating lymphocytes (TILs) are prognostic in different types of cancer. This study aimed to evaluate the prognostic value of PD-1 and PD-L1 expressing TILs in TGCTs.PD-L1 positive TILs were found significantly more often in seminomas (95.9% of patients) and embryonal carcinomas (91.0%) compared to yolk sac tumors (60.0%), choriocarcinomas (54.5%) or teratomas (35.7%) (All p < 0.05). TGCTs patients with high infiltration of PD-L1 positive TILs (HS ≥ 160) had significantly better progression-free survival  and overall survival  opposite to patients with lower expression of PD-L1 (HS < 150). PD-1 expressing TILs were not prognostic in TGCTs.Surgical specimens from 240 patients with primary TGCTs were included into this translational study. The PD-1 and PD-L1 expression on tumor and TILs were detected by immunohistochemistry using anti-PD-1 and anti-PD-L1 monoclonal antibody. Scoring was performed semiquantitatively by weighted histoscore (HS) method.The prognostic value of PD-L1 expressing TILs in TGCTs was demonstrated for the first time. Testicular germ cell tumors (TGCTs) are nearly universally curable malignancies, most commonly found in young men . CisplatMalignant tumor biology and its’ associations with immune mechanisms have been recently a subject of intensified research. It occurs that immune check-point inhibition including PD-1, PD-L1 and/or CTLA4 is a highly effective modality in several types of malignancies –10. Tumo+ T cells, CD8+ T cells, natural killer cells, B cells and monocytes : AB174838) diluted 1:100 in Dako REAL antibody diluent  and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer  for 30 minutes at room temperature, according to the manufacturer's instructions. Color reaction was developed with diaminobenzidine substrate-chromogen solution  for 5 minutes. Finally, the slides were counterstained with haematoxylin, mounted and reacted for 5 minutes with diaminobenzidine substrate-chromogen solution  for visualization. PD-1 and PD-L1 positivity of lymphocytes in the tonsil was used as a positive control, same tissue with omitting of the primary antibody served as negative control.Tissue samples were evaluated under a light microscope , the microphotographs were acquired using the camera  and software DSLR Remote .Tumor cores were independently assessed by two observers (ZC and PB) who were blinded to clinicopathological data. In cases of disagreement, the result was reached by consensus. TILs were identified in hematoxylin-eosin staining according to typical morphology Figure . TILs wiPatients’ characteristics were tabulated. A distri-bution of PD-1 and PD-L1 HS in TILs was significantly different from the normal distribution (Shapiro–Wilk test), therefore we used non-parametric tests for analyses. Analyses of differences in distributions of PD-1 and PD-L1 expression in TILs between the two groups of patients were performed using the Mann–Whitney U test, while Fisher's exact test or the χ2 test when appropriate were used, when PD-1 and PD-L1 expression was categorized as ‘low’ or ‘high’ according to the aforementioned criteria.Median follow-up period was calculated as a median observation time among all patients and among those still alive at the time of their last follow-up. Progression-free survival (PFS) was calculated from the date of orchiectomy or the date of tumor biopsy to the date of progression or death or the date of last adequate follow-up. Overall survival (OS) was calculated from the date of orchiectomy or date of tumor biopsy to the date of death or last follow-up. PFS and OS were estimated using the Kaplan–Meier product limit method and compared by the log-rank test. A multivariable Cox proportional hazards model for PFS and OS was used to assess differences in outcome on the basis of PD1 and PD-L1 expression in tumor cells and TILs of primary tumor and prognosis according to IGCCCG  . All rep