LINE-2A (L2A) endogenous retroelement in the CD274 gene, encoding PD-L1, which causes omission of the transmembrane domain and the regulatory sequence in the canonical 3’ untranslated region. The alternatively spliced CD274-L2A transcript forms the major source of sPD-L1 and is highly conserved in hominids, but lost in mice and a few related species. Importantly, CD274-L2A-encoded sPD-L1 lacks measurable T cell inhibitory activity. Instead, it functions as a receptor antagonist, blocking the inhibitory activity of PD-L1 bound on cellular or exosomal membranes.Immune regulation is a finely balanced process of positive and negative signals. PD-L1 and its receptor PD-1 are critical regulators of autoimmune, antiviral and antitumoural T cell responses. Although the function of its predominant membrane-bound form is well established, the source and biological activity of soluble PD-L1 (sPD-L1) remain incompletely understood. Here, we show that sPD-L1 in human healthy tissues and tumours is produced by exaptation of an intronic  First identified as a marker of developing thymocytes, co-inhibitory receptor programmed cell death protein 1 (PD-1) plays a well-recognised role in restraining T cell responses in autoimmunity, infection or cancer . PD-1 isIn addition to its major membrane-bound form, a soluble form of PD-L1 (sPD-L1) has long been documented in healthy human serum and found elevated in autoimmune disease and in cancer . HoweverCD274 transcript (encoding PD-L1). At least two distinct types of splicing events have been described in several recent reports to remove or affect the exon encoding the PD-L1 transmembrane domain. The first involves mid-exon splicing  to the diversification of the human transcriptome . AbundanCD274 locus comprises four currently RefSeq or GENCODE annotated variants .In an effort to identify aberrant inclusion of EREs in transcripts of cellular genes, we de novo assembled transcripts expressed in a multitude of human cancers, where ERE transcriptional activity is elevated . Togethevariants , each gevariants  for ERE-tive ERE . Transcrintron 6 . TranscrM_A SINE . TranscrCD274-MIRB and CD274-FLAM_A, which include the transmembrane domain encoded by the canonical exon 5, transcript CD274-L2A encodes a truncated isoform of PD-L1, which retains the extracellular Ig-like V and C2 type domains that mediate receptor binding  in intron 4 of the murine Cd274 gene, the major expansion of L2A elements in mammalian genomes is thought to have preceded placental mammal diversification and has now ceased , the equivalent splice donor site in the murine Cd274 gene (GUGAGU) was identical to the intronic part of the consensus splice donor motif GUPuAGU  with the consensus L2A than either the rat or mouse L2A integrations  . However, potential exaptation of the L2A element in the CD274 intron 4 would select for the new function for this L2A element and, consequently, only for the features that are necessary for this new function. These features included the splice site at the end of the CD274 exon 4, the sequence encoding the novel 18 C-terminal amino acids and the polyadenylation site and were provided jointly by the first 29 nucleotides of intron 4 and the first 71 nucleotides of the succeeding L2A element. Indeed, when the sequence divergence of these 100 nucleotides between the splice site at the end of the CD274 exon 4 and the polyadenylation site in the L2A element was examined in 10 primate species, it was found as low as that of coding exons, contrasting the higher divergence of the rest of CD274 intron 4, other CD274 introns and non-coding exons  . This ding exons . These fCD274 variants, variant 1 and CD274-L2A, suggested a degree of co-regulation  or different cancer types (R2 = 0.327)  or lung adenocarcinoma (LUAD) (CD274-L2A (2 = 0.215), with their ratios varying between cell lines by at least two orders of magnitude  . Importaa (LUAD) . Comparaa (LUAD) . For exaD274-L2A . Similaragnitude .CD274-L2A observed here were broadly concordant with those independently reported  were previously used to calculated expression as reads per kilobase per million reads (RPKM) , a modification of RPKM measurements that was developed to eliminate inconsistent calculations across samples  . In cont samples .CD274-L2A expression using variant-specific qRT-PCR at the steady-state, as well as following interferon (IFN) stimulation. To this end, we used a series of B cell leukaemia cell lines, representing progressive steps of B cell differentiation and concomitant PD-L1 expression , generated from human primary leukocyte subsets isolated from peripheral blood of healthy individuals and those with sepsis, Amyotrophic Lateral Sclerosis (ALS), Type 1 Diabetes (T1D), or Multiple Sclerosis (MS) , in comparison with B cells from the same patients, where it was inverted (0.6) . Together, these data highlighted a certain degree of independent regulation of the two transcript variants, as opposed to a fixed rate of aberrant splicing creating the CD274-L2A isoform as a by-product of the canonical variant 1, particularly following IFN stimulation in vitro and in vivo.As copy number, as well as transcriptional regulation of the ukaemias , we nextsis (MS) . Sepsis sis (MS)  and MS pn 1epsis . Express B cells . Both mol levels . In contelevated . As a reCD274-L2A transcript features in hominids, as well as its transcriptional regulation, we first examined the suppressive activity of its protein product. To obtain a source of CD274-L2A-derived sPD-L1 free from exPD-L1 or other potential sPD-L1 forms, we transduced murine B-3T3 cells  and HEK293T cells (which do not express their endogenous CD274 gene) with a CD274-L2A-expressing retroviral vector. Both B-3T3.CD274-L2A and HEK293T.CD274-L2A cell lines produced readily detectable levels of sPD-L1 at 10–40 times higher amounts than those detected by ELISA in supernatants of IFN-γ stimulated HBL-1 cells  production of stimulated CD8+ T cells (CD274 variant 1 (HEK293T.CD274v1), expectedly and significantly suppressed CD8+ T cell activation  exhibited high levels of surface PD-1, assessed by flow cytometry, and correct membrane localisation, assessed by immunofluorescence  or CD274-L2A, which we transfected into HEK293T cells. Transfection with CD274v1 alone increased the surface levels of PD-L1 in a dose-dependent manner, without generating detectable sPD-L1  injections of 200 μg anti-PD-L1  on days 1, 3, 5, and 7 after tumour inoculation.Inbred C57BL/6J (B6) mice were originally obtained from The Jackson Laboratory and subsequently maintained at the Francis Crick Institute’s animal facilities. Eight- to 12-week-old male mice were used for all experiments, randomly assigned to the different treatment groups. All animal experiments were approved by the ethical committee of the Francis Crick Institute and conducted according to local guidelines and UK Home Office regulations under the Animals Scientific Procedures Act 1986 (ASPA) (licence number: PCD77C6D0). For tumour studies, 1 × 10www.repeatmasker.org), configured with nhmmer  program, the Cancer Cell Line Encyclopedia (CCLE), and the indicated individual studies and aligned to our custom transcript assembly, as previously described . Alternaparallel  and Salmparallel . It shouhttps://www.ensembl.org/info/genome/compara/multiple_genome_alignments.html) and with Vector NTI (11.5.0). Sequences were downloaded and plotted with Vector NTI or with AliView (1.21)  . For seq+ T cells isolated by negative selection using a CD8+ T Cell Isolation Kit (Miltenyi Biotec).Peripheral blood was collected from healthy adult volunteers according to protocols approved by the ethics board of The Francis Crick Institute. Peripheral blood mononuclear cells (PBMCs) were freshly isolated by density gradient centrifugation in Ficoll-Paque (VWR) and CD8CD274v1, CD274-L2A, or PDCD1 were generated by adding viral stocks (as described above) to target cells in the presence of polybrene (4 μg/mL) and sorted based on GFP expression to >98% purity on a S3e Cell Sorter (Propel Labs Inc), 72 hr after transduction. Supernatant PD-L1 levels were measured 48 hr after seeding 5 × 105 sorted cells/ml, using the human PD-L1 ELISA Kit  or the murine PD-L1 ELISA Kit , according to manufacturers’ instructions.HEK293T, Jurkat, B-3T3, EL4, MCA-38, CHO, B3, Vero, CV-1 and R9ab cells were obtained from and verified as mycoplasma free by the Cell Services facility at The Francis Crick Institute. Human cell lines were also validated by DNA fingerprinting. All cells were grown in Iscove’s Modified Dulbecco’s Medium (Sigma-Aldrich) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific), L-glutamine , penicillin , and streptomycin . HEK293T, B-3T3, MCA-38, B3 and Jurkat sublines transduced with CD274-L2A, and human PDCD1 were synthesized and cloned into the pRV-GFP vector, constructed and kindly provided by Dr. Gitta Stockinger . Gene synthesis, cloning and mutagenesis were performed by Genewiz LLC and verified by sequencing. Vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped retroviral particles were produced by transfection of HEK293T cells using GeneJuice (EMD Millipore) of vector plasmids with packaging (pHIT60) and VSVg (pcVG-wt) plasmids, kindly provided by Dr. Jonathan Stoye . Virus-containing supernatants were collected 48 hr post-transfection, passed through a 0.45 μm filter and stored at −80°C until further use. Open-reading frames encoding human full-length CD274 or truncated CD274-L2A, and minigenes comprising the full-length CD274 cDNA with an intact intron 4 (CD274 IS4) or an intron 4 lacking the L2A element (CD274 IS4ΔL2A), were additionally synthesized and cloned into the pcDNA3.1 mammalian expression vector for transient transfection experiments.Open-reading frames encoding truncated human or murine-human chimeric Single-cell suspensions were stained for 30 min at room temperature with directly-conjugated antibodies to surface markers. For detection of intracellular antigens subsequent to surface staining, cells were fixed and permeabilised using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following antibodies were used: BV421-labelled mouse PD-L1 , PE-labelled human PD-L1 , APC-labelled human PD-1 , PE-Cy5-labelled or APC Fire 750-labelled human CD25 , PE-labelled human CD69 , APC Annexin V (Biolegend), PerCP-Cy5.5-labelled human CD8 , and APC-labelled human Granzyme B . Multicolour cytometry data were acquired on a LSR Fortessa (BD Biosciences) running BD FACSDiva v8.0 and analysed with FlowJo v10 (Tree Star Inc) analysis software.+ T cells were labelled with Cell Trace Violet (CTV) (Thermo Fisher Scientific) for 20 min and rested overnight before being stimulated for 72 hr with CD3/CD28 Dynabeads (Thermo Fisher Scientific). Jurkat.PDCD1 cells were stimulated for 24 hr with CD3/CD28 Dynabeads (Thermo Fisher Scientific). Primary or cell line T cells were co-cultured with HEK293T cells transfected with pcDNA3.1-CD274v1 or pcDNA3.1-CD274-L2A as indicated. Conditioned media from HEK293T.CD274-L2A cells, parental HEK293T cells, B-3T3.CD274-L2A cells, parental B-3T3 cells, IFN-γ-stimulated HBL-1 cells, parental HBL-1 cells, or 10 μg/mL ultra-LEAF anti-human PD-L1 antibody  were added as indicated.CD85 HEK293T.CD274-L2A cells were grown in the presence of 10 μM GW4869 (Sigma-Aldrich) or DMSO control, or were transfected with varying concentrations of HGS siRNA (Insight Biotechnology). Supernatant was collected after 48 hr and PD-L1 quantified by ELISA according to the manufacturer’s instructions (Abcam). HGS knockdown was assessed by western blot using anti-human HGS monoclonal antibody (Insight Biotechnology) and HRP-conjugated anti-beta actin (Abcam).5 × 10Cell lines were fixed in 4% paraformaldehyde and stained for 30 min at room temperature with PD-1 primary antibody  followed by 1 hr at room temperature with goat anti-mouse Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific) and DAPI (Thermo Fisher Scientific). Cells were mounted in VectaShield mounting medium (Agilent) and imaged on an inverted LSM880 confocal microscope (Carl Zeiss AG).3 cells were seeded in triplicate in a flat-bottom 96-well plate and phase contrast microscopy was used to monitor cell growth on the IncuCyte S3 imaging system (EssenBioScience). Images were collected every 3 hr for 72 hr, and cell proliferation measured using the confluence image mask.6 × 106 cells were stimulated with 100 ng/mL IFN-α or IFN-γ (Abcam) for 48 hr or used untreated. Total RNA from cell lines was isolated using the QIAcube (Qiagen), and cDNA synthesis was carried out with the High Capacity Reverse Transcription Kit (Applied Biosystems) with an added RNase inhibitor (Promega). Purified cDNA was used to quantify CD274v1 and CD274-L2A using variant-specific primers.2 × 10For both variants in all species, the following common forward primer located in a conserved region of exon four was used:TACAGCTGAATTGGTCATCCCA.Common primer | F: The variant-specific reverse primers for human variants were:CD274v1 | R: TCAGTGCTACACCAAGGCATCD274-L2A | R: AGGCAGACATCATGCTAGGTGThe variant-specific reverse primers for African green monkey variants were:CD274v1 | R: TCAGTGCTACACCAAGGAGTCD274-L2A | R: AGGCAGACATCATGCTAGGTGThe variant-specific reverse primers for leporine variants were:CD274v1 | R: TGAATGCTACACCAAGGAACCD274-L2A | R: ATGATGAATATTTACTAGGTGThe variant-specific reverse primers for murine variants were:CD274v1 | R: TGGACACTACAATGAGGAACCD274-L2A | R: GGATGGCCATCGTGCTAGGAAHPRT-specific primers were used in all species:For amplification of a conserved house-keeping gene, the following HPRT | F: TGACACTGGCAAAACAATGCA; R: GGTCCTTTTCACCAGCAAGCTHPRT expression using the ΔCT method.Values were normalised to CD274-L2A in serum-free media. Supernatants from transfected and untransfected cells were centrifuged at 14,000 rpm for 30 min at 4°C and pellets were resuspended in sample loading buffer with or without SDS and 2-mercaptoethanol (β-ME) and heat denaturation. Samples were run on a 4–20% polyacrylamide gel by native or SDS-PAGE, stained with Coomassie Brilliant Blue overnight and visualized on an Amersham Imager 600 .HEK293T, CHO and B3 cells were transiently transfected with pcDNA3.1-t-tests or One Way Analysis of variance (ANOVA) tests. Data that did not pass the variance test were compared with non-parametric two-tailed Mann–Whitney Rank Sum tests or ANOVA on Ranks tests. p values are indicated by asterisks as follows: *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p<0.0001. Hierarchical clustering and heatmap production were performed with Qlucore Omics Explorer 3.3 (Qlucore).Statistical comparisons were made using GraphPad Prism 7 (GraphPad Software) or SigmaPlot 14.0. Parametric comparisons of normally distributed values that satisfied the variance criteria were made by unpaired Student’s  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The PD-1 pathway is an important brake on the immune system. PD-L1 is a cell surface ligand that activates PD-1 on T cells, and both proteins are key targets in cancer immunotherapy. Kassiotis et al. report that the acquisition of a jumping gene in the PD-L1 gene created a shortened isoform of PD-L1. This new isoform is soluble and antagonize the function of full length PD-L1 in cells and in animals. This ancient genetic insertion is retained in many species but lost in others, providing an a compelling example of how the evolutionary battle between mammalian genomes and jumping genes diversifies and reshapes immunity.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Howard Y Change as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Satyajit Rath as the Senior Editor.Thank you for submitting your article "Soluble PD-L1 generated by endogenous retroelement exaptation is a receptor antagonist" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The authors revealed that the exaptation of endogenous retroelement Line-2A in PD-L1 generates alternatively spliced variant as a soluble PD-L1 (sPD-L1). They further characterized the function of this sPD-L1 and identified a role of sPD-L1 in blocking the inhibitory activity of membrane-bound PD-L1.Essential revisions:1) The presence of a soluble PD-L1 isoform (sPD-L1) is known. The novelty of this work is the recognition that the presence of intronic Line-2A produces the soluble PD-L1 by omitting splicing site at the end of exon4 and providing the terminal exon and polyA. However, this conclusion lacks convincing evidence. To confirm the role of Line-2A in alternative splicing of PD-L1, it would be essential to delete intronic Line-2A in PD-L1 to test if the splicing variant of sPD-L1 is decreased. Hassounah et al., 2019) showed that this soluble PD-L1 is generated by the integration of HPV at the intron after exon 4 which is located in Line-2A region. Given that a loss of sPD-L1 variant in mice and the difference of Line-2A between mice and human, it would be better to map the key segment of Line-2A that controls the alternative splicing to distinguish if this effect is from HPV integration site or other sites in Line-2A. In addition, in Figure 1F, the authors showed gene tracks of different splicing variant. It would be better to design qPCR primer specifically targeting the terminal exon provided by Line-2A to confirm this sPD-L1 variant.2) The authors claim that the L2A was exapted for a beneficial function. However, a much more rigorous evolutionary analysis is necessary to support this idea. For example, in Figure 2F, the authors conclude that the higher sequence similarity of the hominid L2A insertion to the consensus L2A reflects purifying selection on that element in hominids. But, this could also reflect a globally faster mutation rate in rodents, rather than purifying selection at this specific insertion. This evolutionary analysis must be substantially improved to support the idea this insertion was exapted, by including other L2A elements including ones expected to be neutrally evolving, and other nearby intronic sites. Furthermore, while the authors state that canonical splice/polyA sites are conserved in other species at the sequence level, it would be much more convincing if there were evidence that the sPD-L1 transcript is indeed produced in neutrophils in other species.3) However, they include no in vivo studies to support these results. This is important to interpret the significance of sPD-L1. For example, an experiment examining administration of sPD-L1 in tumor bearing mice (or some other model) alone and in combination with anti-PD-1 mAb. Further, it would be interesting to see the effect of combination with anti-PD-L1 mAb – as the authors report, there has been a recent study showing sPD-L1 may interfere with the effect of anti-PD-L1 immunotherapy. These experiments would demonstrate the importance of sPD-L1 in the PD-L1 – PD-1 axis in immunity and facilitate therapeutic application. Overall, this paper offers some novel insight for basic biology and its potential importance in immunity and therapy. But unfortunately, they did not demonstrate its relevance for normal function or therapy in this paper.4) In Figure 4-5, the authors characterized the function of sPD-L1 by overexpressing of this sPD-L1 variant. To test the role of endogenous sPD-L1 in T cell inhibition, it would be better to use a cell line that has highly expressed sPD-L1  and delete the Line-2A as a control to compare the T cell inhibition. In addition, several studies showed that sPD-L1 has a T cell inhibitory activity, which is opposite to the author's findings. Although the authors suggested that the discrepancy is due to artificial Fc fusion in those studies, Mahoney et al., 2019) showed the T cell inhibitory activity of sPD-L1 without using Fc fusion. Thus it is essential to test the function of endogenous sPD-L1 to avoid the discrepancy and highlight the physiological function of sPD-L1.5) As the authors mentioned, many of the reported experiments for CD274-L2A have been performed previously in the cited Hassounah et al., 2019 and Mahoney et al., 2019, including the TCGA and CCLE transcriptome analysis, whether it inhibits T cell function, and whether it binds to PD-1. The authors find some contradictory results compared to published studies, and did a good job discussing the possible reasons for the inconsistency. However, the authors should perform more experiments to substantiate these differences, as these differences are crucial for the novelty of the study. Examples: a) The authors reported weak correlation between expressions of CD274-L2A and the full-length variant 1 in both healthy tissues and different TCGA cancers in Figure 3A, which is inconsistent with previous results. The authors mentioned that such difference is likely due to different RNA-seq quantitation methods, but they should clearly state what's the difference, and why their method is better. The correlation analysis should be performed at individual level within each tissue, rather than being performed using the averages of different tissue types, which is apparently depicted in Figure 3A.b) Figure 4B showed monomeric sPD-L1 at ~28 kDa, and a tetramer  The presence of a soluble PD-L1 isoform (sPD-L1) is known. The novelty of this work is the recognition that the presence of intronic Line-2A produces the soluble PD-L1 by omitting splicing site at the end of exon4 and providing the terminal exon and polyA. However, this conclusion lacks convincing evidence. To confirm the role of Line-2A in alternative splicing of PD-L1, it would be essential to delete intronic Line-2A in PD-L1 to test if the splicing variant of sPD-L1 is decreased. Hassounah et al., 2019) showed that this soluble PD-L1 is generated by the integration of HPV at the intron after exon 4 which is located in Line-2A region. Given that a loss of sPD-L1 variant in mice and the difference of Line-2A between mice and human, it would be better to map the key segment of Line-2A that controls the alternative splicing to distinguish if this effect is from HPV integration site or other sites in Line-2A. In addition, in Figure 1F, the authors showed gene tracks of different splicing variant. It would be better to design qPCR primer specifically targeting the terminal exon provided by Line-2A to confirm this sPD-L1 variant.We have now included experiments where we have specifically deleted this intronic L2A element as the reviewer suggests. To this end, we constructed a CD274 minigene, incorporating either the entire intron 4, which contains the exonised L2A, or an intron 4 with the L2A deleted. Introduction of the reference intron 4 to the CD274 cDNA restored production of sPD-L1 protein, whereas deletion specifically of the L2A element in this minigene abolished this production. These results establish the critical requirement for the L2A element in the production of sPD-L1 and are shown in the new Figure 1—figure supplement 2A-C. The requirement for the L2A element is also supported by new data on sPD-L1 production by cells from other species, according to the integrity of the L2A and splice and polyadenylation sites .The reviewer might have misinterpreted the study by Hassounah et al. We should clarify that an integration of HPV16 was previously found in intron 4 of the CD274 gene in a single case of head and neck squamous cell carcinoma . This was a somatic, likely extrachromosomal event, where the entire CD274 gene, HPV16 insertion and part of the upstream PLGRKT gene underwent structural rearrangement, amplification, and overexpression.It was this finding that inspired Hassounah et al. to look for novel variants transcribed from the CD274 locus, which allowed then to independently identify the CD274-L2A transcript. It is also interesting to note that our inspection of this somatic HPV16 integration shows that it happened right in the middle of the L2A element, replacing its function and highlighting its importance. We must reiterate, however, that the HPV16 integration is a somatic event in a single tumour and no HPV integration exists in the human genome, nor any effect of HPV can be expected. In contrast, the L2A integration is fixed in the human germline  and is essential for the production of the CD274-L2A transcript and of the sPD-L1 protein in unmutated cancers and healthy tissues.We had indeed verified and quantified expression of the CD274-L2A transcript in human cells by qRT-PCR using primers specific to this transcript (with the reverse primer complementary to the L2A element). These results were shown in the original Figure 3C. We have now included similar qRT-PCR validation in non-human primate cells .2) The authors claim that the L2A was exapted for a beneficial function. However, a much more rigorous evolutionary analysis is necessary to support this idea. For example, in Figure 2F, the authors conclude that the higher sequence similarity of the hominid L2A insertion to the consensus L2A reflects purifying selection on that element in hominids. But, this could also reflect a globally faster mutation rate in rodents, rather than purifying selection at this specific insertion. This evolutionary analysis must be substantially improved to support the idea this insertion was exapted, by including other L2A elements including ones expected to be neutrally evolving, and other nearby intronic sites. Furthermore, while the authors state that canonical splice/polyA sites are conserved in other species at the sequence level, it would be much more convincing if there were evidence that the sPD-L1 transcript is indeed produced in neutrophils in other species.We agree with the reviewer and appreciate the suggestions. Higher apparent conservation of the L2A in humans than in mice does not prove purifying selection in the former and it is, as the reviewer points out, more likely caused by faster evolution of mice. It is nevertheless interesting to note that the mutations in rodents affect specifically the production of CD274-L2A.CD274 intron 4, other CD274 introns and non-coding exons.Overall, the L2A fragment in the CD274 seems to be evolving at an average rate in humans . However, as indicated by original alignment in Figure 2A-B, this part of intron 4 appears as well conserved in hominids as the coding exons. We have now extended this analysis over the entire CD274 locus , to show that in 10 primate species, the 100 nucleotides covering the exonised part of intron 4 and embedded L2A element were as conserved as the coding exons, contrasting with the rest of Moreover, following the reviewer’s suggestion, we have now examined whether or not the CD274-L2A transcript is produced as predicted by the splice and polyadenylation sequences in other species. As primary neutrophils (some of the shortest-lived cells of the body) from other species are not available, we used green monkey cells lines (CV-1 and Vero), rabbit cell line (R9ab) and mouse cell lines (EL4 and MCA-38), all of which express the respective full-length CD274. However, whereas CD274-L2A was readily detectable in non-human primate cells, it was absent from rabbit or mouse cells, as predicted by splice site and L2A sequences.3) However, they include no in vivo studies to support these results. This is important to interpret the significance of sPD-L1. For example, an experiment examining administration of sPD-L1 in tumor bearing mice (or some other model) alone and in combination with anti-PD-1 mAb. Further, it would be interesting to see the effect of combination with anti-PD-L1 mAb – as the authors report, there has been a recent study showing sPD-L1 may interfere with the effect of anti-PD-L1 immunotherapy. These experiments would demonstrate the importance of sPD-L1 in the PD-L1 – PD-1 axis in immunity and facilitate therapeutic application. Overall, this paper offers some novel insight for basic biology and its potential importance in immunity and therapy. But unfortunately, they did not demonstrate its relevance for normal function or therapy in this paper.As the reviewer suggested, we have now investigated the role of sPD-L1 in an in vivo tumour setting using the MCA-38 colon adenocarcinoma cell line, which has been described to be sensitive to PD-1/PD-L1 blockade. MCA-38 cells expressing human or murine sPD-L1 phenocopied the delayed tumour growth with exogenous PD-L1 blockade when subcutaneously transplanted into immunocompetent mice despite having equivalent in vitro growth rates. These results are displayed in Figures 6A-B and Figure 6—figure supplement 1. Whilst these experiments support in vivo biological activity for sPD-L1 in tumour bearing mice, consistent with our in vitro data, we do acknowledge that this is one of many settings in which the antagonistic activity of sPD-L1 could be important. To fully elucidate the physiological function of sPL1 beyond the tumour setting, we would have to study a mouse model and, as mice naturally lack production of sPD-L1, we would first have to restore it in by re-introducing the ancestral intron 4 into the mouse germline. This would allow the study of the physiological function of sPD-L1  in processes, such as immune development, autoimmunity, viral infection or placentation, where preliminary results suggest it might play an important role. However, these experiments are far beyond the scope of the current manuscript.4) In Figure 4-5, the authors characterized the function of sPD-L1 by overexpressing of this sPD-L1 variant. To test the role of endogenous sPD-L1 in T cell inhibition, it would be better to use a cell line that has highly expressed sPD-L1  and delete the Line-2A as a control to compare the T cell inhibition. In addition, several studies showed that sPD-L1 has a T cell inhibitory activity, which is opposite to the author's findings. Although the authors suggested that the discrepancy is due to artificial Fc fusion in those studies, Mahoney et al., 2019) showed the T cell inhibitory activity of sPD-L1 without using Fc fusion. Thus it is essential to test the function of endogenous sPD-L1 to avoid the discrepancy and highlight the physiological function of sPD-L1.Although we produce sPD-L1 by expression in cell lines that do not normally express it, we should note that we have used it at more physiological levels and ratio with the full-length form than the previous studies. For their immunosuppression assays, Mahoney et al. used a recombinant purified form of a HIS-tagged, rather than an Fc-fusion of sPD-L1, and reported only weak suppression at concentrations between 10 and 20 micrograms/ml. We have used native sPD-L1 at 2 nanograms/ml  or lower and in par with the physiological concentration of sPD-L1. At these levels, we have consistently failed to detect suppressive activity, whereas antagonistic activity was still readily measurable. We should also note that the cell lines with high expression of sPD-L1 do not necessarily reflect physiology. For example, B cell leukaemias and derived cell lines (like Ramos or HBL-1), very frequently exhibit extensive structural rearrangement and amplification of the CD274 locus . This not only affects the physiological ratio of membrane-bound, exosomal and soluble PD-L1 (as part of the transformation process), but also creates the practical problem of correct targeting of the various rearranged and amplified CD274 copies. Consequently, our effort to target the endogenous CD274 locus in such cell lines have been thwarted by its rearrangements and amplification. Complete elucidation of the physiological function of sPD-L1 will therefore necessitate the creation of a mouse strain, where the ancestral allele of Cd274 is restored. As we outlined in our reply to comment 3 above, this would be the only way to study of the physiological function of sPD-L1  in processes, such as immune development, autoimmunity, viral infection or placentation.5) As the authors mentioned, many of the reported experiments for CD274-L2A have been performed previously in the cited Hassounah et al., 2019 and Mahoney et al., 2019, including the TCGA and CCLE transcriptome analysis, whether it inhibits T cell function, and whether it binds to PD-1. The authors find some contradictory results compared to published studies, and did a good job discussing the possible reasons for the inconsistency. However, the authors should perform more experiments to substantiate these differences, as these differences are crucial for the novelty of the study. Examples: a) The authors reported weak correlation between expressions of CD274-L2A and the full-length variant 1 in both healthy tissues and different TCGA cancers in Figure 3A, which is inconsistent with previous results. The authors mentioned that such difference is likely due to different RNA-seq quantitation methods, but they should clearly state what's the difference, and why their method is better. The correlation analysis should be performed at individual level within each tissue, rather than being performed using the averages of different tissue types, which is apparently depicted in Figure 3A.b) Figure 4B showed monomeric sPD-L1 at ~28 kDa, and a tetramer  and retains receptor binding, its expression is detected in healthy tissues and it is elevated in several cancer types. The single most important difference is that we find that CD274-L2A-encoded sPD-L1 is a receptor antagonist, a possibility that was not examined in the previous studies.There are, however, other methodological differences that the reviewer perceptively identified:CD274 locus and normalised expression by the total transcript count . We have now added the new Figure 3—figure supplement 1, depicting the correlation performed at individual level within each tissue, as per reviewer’s suggestion. We focused on tissues/cancers with sufficient expression and number of samples to enhance our ability to detect even weak correlations. The new analysis corroborates our earlier analysis, in that there seems to be little correlation between expression of the two variants. For example, healthy lung expresses primarily the full-length isoform, whereas in LUAD the balance shifts in favour of the truncated isoform.a) Indeed, our analysis of RNA-seq data from a multiple cancer types and healthy tissues indicates only weak correlation between the expression of full-length and truncated PD-L1 variants. We do observe a correlation in the same direction as the previous two studies, simply not as strong and certainly not as statistically significant. There are several differences, which are now discussed in the text. In their analysis, Mahoney et al. used a non-standard method for quantitation of the two isoforms based of the number of reads mapping uniquely to the parts of the two isoforms that are different, and normalised by the read count in the RNA-seq library (a variant of RPKM). Similarly, Hassounah et al. used the ratio of exon 4 (shared exon) to exon 5 (not present in CD274-L2A) as a proxy for CD274-L2A expression, which they calculated again using the RPKM method. Instead, we used the TPM method, originally proposed as an alternative to RPKM due to inaccuracy in RPKM measurements . Also, using our extended assembly, we took into account any other exon-sharing transcript produced by the b) We agree with the reviewer that our original Western blots were low quality. This is due to the poor ability of the available antibodies to detect the native form of PD-L1. We have tested all widely-used PD-L1 clones suitable for immunoblot , and none showed cleaner bands under native conditions. Of note, Hassounah et al. use clone 28-8 (Abcam) only under denaturing conditions and did not show any data under native conditions. Mahoney et al. showed data under native conditions, but used an in-house clone 368A.5A4 that is not commercially available and could not be tested here. As far as we can tell, this is the only clone reported to detect PD-L1 under native conditions.To conclusively determine the molecular form of native sPD-L1, without the need for an antibody compatible with native Western blot, we have now expressed native sPD-L1 (without any tags or Fc fusions), in three separate cell lines (including those used in the previous studies), which we grew in protein-free media. This allowed us to directly and clearly visualise native, as well as denatured sPD-L1 by Coomassie stain of native and denaturing PAGE gels. These results are now shown in the modified Figure 4B and support our previous conclusion based on Western blotting, revealing that the native form of sPD-L1 has a molecular weight consistent with a tetramer (~120 kDa) and the monomeric form a molecular weight of ~30 kDa, close to its theoretical weight of 28 kDa.


Mono-, few-, and multilayer graphene is explored towards the electrochemical Hydrogen Evolution Reaction (HER). Careful physicochemical characterisation is undertaken during electrochemical perturbation revealing that the integrity of graphene is structurally compromised. Electrochemical perturbation, in the form of electrochemical potential scanning (linear sweep voltammetry), as induced when exploring the HER using monolayer graphene, creates defects upon the basal plane surface that increases the coverage of edge plane sites/defects resulting in an increase in the electrochemical reversibility of the HER process. This process of improved HER performance occurs up to a threshold, where substantial break-up of the basal sheet occurs, after which the electrochemical response decreases; this is due to the destruction of the sheet integrity and lack of electrical conductive pathways. Importantly, the severity of these changes is structurally dependent on the graphene variant utilised. This work indicates that multilayer graphene has more potential as an electrochemical platform for the HER, rather than that of mono- and few-layer graphene. There is huge potential for this knowledge to be usefully exploited within the energy sector and beyond. The production of molecular hydrogen due to the electrocatalytic splitting of water via the Hydrogen Evolution Reaction  is well-known and improving the overall process, output and costs can give rise to a desirable future source of sustainable energy4.The electrocatalytic splitting of water is considered a promising strategy for the production of hydrogen, which is a clean and carbon neutral fuel with potential applicability in a range of commercial, industrial and transportation sectors7. However, Pt is a precious metal with a low natural abundance in the Earth’s crust and has a prohibitive cost for wide-spread implementation in water electrolysers8, meaning that for this technology to prevail, an alternative cheaper yet still efficient catalyst is required. As a result of their beneficial properties 13, there is interest in the application of 2D materials, such as graphene17, graphene oxide18, and other (non)carbon nanomaterials19 to be explored as Pt alternatives.Currently, platinum (Pt) is considered the most active catalyst towards the HER given that it has a small binding energy for the reaction to occur, resulting in the reaction proceeding at low over-potential values close to zeroet al.20 have reviewed the use of pristine, doped and hybrid graphene materials for the electrocatalytic splitting of water, indicating that graphene has been subjected to substantial investigation and thus implemented to undertake multiple roles within this area, such as being the electrochemical platform and/or as a functionalizable support29 for use within a multitude of distinct carbon-based hybrid catalysts. That said, there is a lack of research and understanding with respect to the application of graphene towards the HER in terms of the graphene structure and how the surface changes as a function of electrochemical perturbation.For example, Qu This paper fully characterises, for the first time, the electrochemical performance of mono-, few- and multilayer graphene electrodes towards the HER, with the purpose of correlating the observed electrochemical behaviour to the change in the physical structure of the graphene surfaces using electrochemical perturbation in the form of electrochemical scanning (linear sweep voltammetry) and Raman analysis.2SO4), as is common within the literature31; which is the cathode in polymer electrolyte membrane (PEM) electrolysers. A bespoke electrochemistry 3D printed cell was developed and utilised . The physicochemical characterisation of the graphene sample (and others) are presented in the SI, confirming a true monolayer graphene surface. Figure ca. −0.669 V, a current density at −0.65 V of −7.39 mA cm−2 and a Tafel slope of 234 mV dec−1 , indicating a reduced electrochemical activity. The HER is less electrochemically reversible than conventional Pt systems (30 mV dec−1), which is as expected due to Pt being a pure metal that has a very small binding energy for H+ 32; whereas pristine graphene’s surface is mostly comprised of basal planes, that are reported to have limited electrochemical activity36.Attention was first turned to benchmarking monolayer graphene, without any further modification from the manufacturer, as an electrochemical platform towards the HER within acidic media , the region in which the HER occurs; this type of stability study is often overlooked with the academic literature. Additionally, after each scan, the graphene sample was interrogated with Raman to allow the direct comparison of electrochemical perturbation upon the graphene’s physical structure. Surprisingly, after five successive LSV scans, the current density at −0.65 V was −1.60 mA cm−2 with an onset potential of ca. −0.859 V and a Tafel slope of 280.5 mV dec−1 (vs. RHE), showing a decrease in the electrochemical HER performance. Interestingly, after electrochemical perturbation of ten voltammetric scans, an onset value of ca. −0.59 V and a current density at −0.65 V of −15.11 mA cm−2 is evident, indicating an improvement in the onset potential, however the Tafel slope had changed to 292.0 mV dec−1. Furthermore, after a further ten LSV scans , an onset value of ca. −0.099 V and a limited current density at −0.85 V of −0.0014 mA cm−2 was exhibited, with a Tafel slope of 141.1 mV dec−1 describing poor electrochemical activity throughout the HER experiments, indicating an overall significant drop in the electrochemical current and reduced electrochemical activity. These changing electrochemical observations clearly suggest a physical change of the monolayer graphene and in the conductive pathways. In summary, the monolayer graphene initially gives rise to a useful electroactive graphene surface, which upon electrochemical perturbation and potential cycling ultimately reduces to a worse electrochemical response towards the HER.Critically, the effect of electrochemical perturbation, in terms of electrochemical reversibility, stability and cyclability was explored after each LSV scan. Figure 2) reported above. Notably, after twenty LSV scans, there is no electrochemical response and as depicted in Fig. In order to understand this phenomenon, Raman mapping of the monolayer graphene surface was explored C0) calculations are now explored in order to estimate the edge and basal plane coverages of respective ‘pristine’ and ‘damaged’ graphene samples. Total specific capacitance is calculated as described by Eq. Ce) and basal plane (Cb) contributions comprising the graphene surface37:−2) are the specific weighted capacitance averages for edge and basal plane surfaces respectively and basal plane of highly ordered pyrolytic graphite (HOPG)38 have reported specific capacitance values of ca. 1–2 µF cm−2, while the specific capacitance of edge plane orientated HOPG is ca. 70 µF cm−2, allowing one to estimate via Eq.  is presented in Table 2 bubbles are being generated. Growing bubbles (due to the HER process) can act as collectors of smaller ones, inducing mechanical forces and mass diffusion43, that are likely to be responsible of the generation of the first rips on the graphene surface. As shown in Fig. 44. Figure ca. 1590 cm−1 and 2690 cm−1 respectively45, instead only background noise is present due to the SiO2/Si wafer onto which the graphene was supported. Given the above insights, the observed initial improvement in the electrochemical performance of monolayer graphene is most likely due to an increase in the planar edge density when a hole is created and the surface is ripped , which leads to an increase in electrochemical behaviour at first , hence there is a reduction in the overall electrochemical behaviour or even the full destruction of the graphene monolayer. Note that it has been previously reported that Chemical Vapour Deposition (CVD) grown graphene and SiO2 substrates have different wettability properties, although underlying substrates do not seem to affect the wettability of such graphene47; in the presence of graphene with rips and holes as this manuscript reports, is likely that the different areas might have different wettability, such effect needs further exploration and will be investigated in the future.The behaviour observed in Figs In order to gain further insights, it is interesting to compare the above results with few- and multilayered graphene, applying the same analysis of stability and cyclability experiments towards the HER. Thus, we next investigate if the thickness of graphene layers has an impact on the structural integrity.−2 to −0.0042 mA cm−2. In contrast, the multilayer graphene sheet increased its current density from −0.028 mA cm−2 to −0.41 mA cm−2 when scanned one hundred times. Evidently, the surface of few-layer graphene is susceptible to similar surface changes as observed with monolayer graphene (but to a less significant degree than the latter), whereas multilayered graphene remains unaltered and stable throughout experiments (with an intrinsic resistance to such surface changes apparent).Few- and multilayer graphene electrodes were explored towards the HER , creating surface defects due to cavitational forces and a large edge plane % (i.e. causing an observed improved response), after which ultimately the integrity of the graphene film as a whole is not viable and the electrical conductive pathways are disrupted and result in a loss of electrochemical signal. This response is mirrored with few-layered graphene structures, but not to the same extent. Conversely, multilayered graphene structures do not present this phenomena and remain stable  after HER scanning for an extensive number of scans. These findings are of high importance to those working in the graphene energy field, particularly for those designing and implementing graphene electrical components given that the sheet integrity is questioned herein and this report shows that pristine graphene is not a beneficial electrode material towards the HER.In summary, this paper indicates that mono- and few-layer graphene, when used as electrodes toward the HER, break-up when an electrical current is applied to them during electrochemical HER experiments performed within aqueous solution. The mechanism of which is first ripping of the film due to the evolution of H2SO4.All chemicals used were analytical grade and were used as received from the supplier  without any further purification. All solutions were prepared with deionised water of resistivity no less than 18.2 MΩ cm and were vigorously degassed prior to electrochemical measurements with high purity, oxygen free nitrogen. The tested solutions were 0.5 M Hquasi-graphene) and multilayer-graphene films supported on an oxidised silicon wafer. A Pt wire counter/auxiliary electrode and a silver/silver chloride  reference electrode completed the circuit.Electrochemical measurements were performed using a three-electrode system on an Autolab PGSTAT204 potentiostat . Working electrodes were: commercially obtained chemical vapour deposition (CVD) synthesised mono-layer, a few-layer 50 and are known as ‘graphene on 285 nm SiO2 Wafer’ and have been previously reported and characterised in the literature53; the exact details are proprietary information50. Note that full physicochemical characterisation (Raman spectroscopy and X-ray photoelectron spectroscopy (XPS)) of the various graphene samples utilised within this work is reported in the Supplementary Information.The commercially available CVD synthesised graphene films, that have been used in our previous workThe graphene working electrode was secured into a bespoke 3D printed electrochemical cell that is described fully in the Supplementary Information Fig.  and is c−1 was employed. Raman maps show intensity of graphene’s G band  in order to show the presence of graphene. It is important to note that it has not been detected the presence of D or D’ Raman band in the samples, which is likely to be due to the large sample size  with newly-created defects in the macroscale. In this case, it is only detected the presence of graphene (in different number of layers) or the lack of graphene presence (lack of graphene Raman peaks with only SiO2 Raman peaks present).Raman Mapping Spectroscopy data was performed using a DXR Raman Microscope  fitted with a 532 nm excitation laser at a low power of 3 mW to avoid any heating effects. Spectra were recorded using a 3 seconds exposure time for 3 accumulations at each point. To collect the map a step size of 75 × 75 µm and a Raman profile between the region of 1050 and 3300 cm27:The Tafel analysis method is utilised in the academic literature to describe the underlying electrochemical processes of the HER, and are an indicator of the reaction mechanism. The Volmer-Heyrovsky-Tafel mechanism includes+ discharge step being the Volmer reaction, leading to the following equation29:F is the Faraday constant, R is the universal gas constant and T is the temperature at which the electrochemical experiment was performed, each step is capable of being the rate-determining step of the HER. This analysis is dependent on the corresponding Tafel slope.the initial H−2 from the plot of log current density vs. potential values.The Tafel Slope and onset values have been calculated at 10 mA cmSupplementary information


Overexpression of the MUC5B protein is associated with idiopathic pulmonary fibrosis (IPF), but little information is available regarding the pathogenic effects and regulatory mechanisms of overexpressed MUC5B in IPF. MUC5B, with MUC5B promoter polymorphism, MUC5B gene epigenetic changes, effects of some transcriptional factors, and inflammatory mediators in IPF. In addition, the associated signaling pathways which may influence the expression of MUC5B are also discussed. The overexpression of MUC5B in terminal bronchi and honeycomb cysts produces mucosal host defensive dysfunction in the distal airway which may play an important role in the development of IPF. This review addresses the possible association of overexpression of This work has important implications for further exploration of the mechanisms of overexpression of MUC5B in IPF, and future personalized treatment. Idiopathic pulmonary fibrosis (IPF), a lung disease of unknown etiology, is characterized by progressive lung scarring of the pulmonary parenchyma and the formation of subpleural honeycomb cysts. The published incidence of IPF has ranged from 0.6 per 100,000 person years to 17.4 per 100,000 person years in different areas from 1981 to 2009 . HoweverTERT), telomerase RNA component (TERC), poly(A)-specific ribonuclease (PARN), and regulator of telomere length elongation helicase (RTEL) are implicated in the maintenance of telomere length, and surfactant protein C (SPC) and surfactant protein A2 (SPA2) have been recognized to maintain surfactant function [MUC5B), desmoplakin (DSP), phospholipid transporting 11A ATPase, and toll interacting protein (TOLLIP), which play important roles in the maintenance of bronchoalveolar epithelium function and immune regulation [IPF has a known genetic component, as evidenced by the fact that as many as 1/5 of affected individuals report having a family member with pulmonary fibrosis . Geneticfunction –11. Besifunction  have idegulation . MUC5B gene (rs35705950) that is the principal risk factor (genetic and otherwise) for developing IPF, accounting for 30–35% of the risk [Up to now, extensive genome-wide linkage scanning has identified a single nucleotide polymorphism (SNP) in the promoter region of thethe risk , 12–19.MUC5B protein may play a direct role in the pathogenesis of IPF regardless of the allele status . The ideIn the normal lungs, mucus is responsible for trapping inhaled particles, including bacteria, and transporting them out of the airways by ciliary and cough-driven forces. Meanwhile, mucus also helps remove endogenous debris including dying epithelial cells and leukocytes , 22. MucMUC5AC and MUC5B are expressed throughout the upper and lower respiratory tract. In the tracheobronchial conducting airways, both mucins are synthesized by surface goblet cells and submucosal glandular cells. It has been confirmed in Seibold et al.'s study  that theIPF has always been considered to primarily involve the alveolar region . Howeverβ, MMP-9, and MMP-7 in the bronchiolar epithelium in UIP patients is also provided by this study. In the perspective of imaging features, it is suggested that, in the earliest stages of IPF development, patients have the increased measures of an internal perimeter of 10mm (Pi10) which are commonly viewed as increased measures of airway wall thickness [Except for the aberrant physiologic alterations  which are consistent with distal airways abnormalities in IPF , earlierhickness . Taken t MUC5B gene, the intron 5 variant rs2076295 in desmoplakin (DSP) gene, and their enhanced expression localized to the distal airway epithelial cells are considered associated with IPF pathogenesis [Besides the histological studies, a growing body of evidence from genetics has implicated biologic processes occurring in the distal airways as potential drivers of IPF pathogenesis. For example, the promoter SNP rs35705950 inogenesis –32. In t+ / MUC5AC+ distal airways in IPF patients are both significantly greater than those in normal subjects, and the MUC5B+ distal airways are especially more frequent. Perhaps the conversion of MUC5B− distal airways to MUC5B+ airways and the increase of cells expressing MUC5B in distal airways occur in IPF subjects.In Seibold et al.'s study , dual imIt is well established that the characteristic changes of HRCT in IPF/UIP subjects are always subpleural honeycomb cysts and lower zone predominant reticular infiltrates. However, there exists another form of honeycomb cysts in IPF, namely, microscopic honeycomb, which does not correlate with the honeycomb observed in HRCT representing dilated airways due to traction from the fibrotic process. Epithelial cells expressing MUC5B are the dominant mucin-expressing cell type in microscopic honeycomb cysts. The microscopic honeycomb cysts are filled with MUC5B protein and chronic inflammation cells and lined by pseudostratified ciliated columnar epithelium similar to those which line the bronchioles , 33, 34.Mucus overproduction contributes to the morbidity of many airway diseases, among which the most common are chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis (CF), and diffuse chronic panbronchiolitis –43. In aFirst, excessive MUC5B compromises the mucosal host defense and reduces lung clearance of inhaled particles, dissolved chemicals, and microorganisms. Over time, reduced clearance may lead to scar tissue formation and persistent fibroproliferation that expands and displaces normal lung tissue. Given that cigarette smoking is a strong risk factor for the development of IPF , it is lSecond, excessive MUC5B in the respiratory bronchioles may interfere with alveolar repair. It has been established that local expansion of type II alveolar epithelial cells following lung injury may repopulate denuded alveolar basement membranes , 50. OneThe changes of MUC5B in the distal conducting airways potentially enhance injury or disrupt repair responses in alveoli. The two mechanisms are plausible and may act alone or together to contribute to the development of idiopathic pulmonary fibrosis. MUC5B gene in pulmonary diseases will contribute to the exploration of explicit signaling pathways in MUC5B overproduction. At present, genetic mutation, epigenetic changes, effects of some transcriptional factors as well as inflammatory mediators, and the associated signaling pathways may be involved in the regulation of MUC5B production.The overproduction of MUC5B plays an important role in the development of IPF; however, little is known about the influencing factors of gene expression. Identifying the associated influencing factors of the MUC5B promoter, within 4 kb of the MUC5B transcription start site. An SNP, rs35705950, is a G to T transversion that occurs in an area of the MUC5B 5'flanking region, 3 kb upstream of the transcription start site, exhibited the strongest association with both familial interstitial pneumonia and idiopathic pulmonary fibrosis. In a survey conducted in a European Caucasian population, the T risk allele was present in 41.9% of the IPF patients and 10.8% of the controls [ MUC5B promoter is still significantly associated with the risk for IPF.From allelic testing, Seibold et al.  observedcontrols , while icontrols . Althoug MUC5B is strongly associated with the development of IPF, there are still many individuals with this SNP rs35705950 that do not develop to IPF [ MUC5B gene, thus changing the expression of MUC5B. Epigenetic changes, such as CpG motif methylation, belong to the non-Mendelian genetics, but are heritable and always influenced by the environmental factors [ MUC5B promoter may establish a link between environmental exposures and the MUC5B promoter polymorphism in IPF.Although the polymorphism in the promoter ofp to IPF , 19. Thep to IPF , cigaretp to IPF , and somp to IPF  such as  factors . Therefo MUC5B promoter variant rs35705950 has been validated as a contributor to the expression of MUC5B in the lung [ MUC5B promoter contributes to the expression of MUC5B in IPF. This polymorphism may be related to the methylation in the MUC5B promoter region and the disruption of the activities of some transcriptional factors.Many studies show that thethe lung , 56, 57. MUC5B promoter region showed that MUC5B was highly sensitive to DNA methylation [ MUC5B promoter variant (Chr11: 1240989–1241950), MUC5B expression, and IPF. This region is hypermethylated in association with IPF, increased expression of MUC5B in lung tissue, and the rs35705950 risk allele (T). These findings define a regulatory region and support a role for DNA hypermethylation in the regulation of MUC5B expression in the lung and as a risk factor for the development of IPF.Previous epigenetic analysis of thehylation . Hellinghylation  further  SPC and SPA2 have been described in association with FIP and rarely with sporadic IPF. Considering that the MUC5B promoter variant rs35705950 is associated with adult IPF, Liptzin et al. [ SPC mutations. The study showed that MUC5B was increased in BALF and lung tissues of pediatric IPF patients with SPC mutations compared with the controls, indicating that MUC5B may play a role in the development of IPF in patients with SPC mutations. Previous studies suggested that the mutations in SPC could lead to endoplasmic reticulum (ER) stress [As previously mentioned, mutations in the genes forn et al.  examined) stress –63 which MUC5B rs35705950, genome-wide association studies (GWAS) have identified other polymorphisms conferring risks to IPF. Among these loci, rs2736100 in TERT genes were also identified in several independent studies [ TERT and MUC5B polymorphisms independently and differentially confer susceptibility to IPF.Besides studies , 64. Wei studies  tested t MUC5B gene. The study conducted by Noth et al. [ TOLLIP and MUC5B are in weak linkage disequilibrium, and the association of TOLLIP genetic variants with IPF susceptibility is independent from that of the MUC5B promoter SNP. However, whether the MUC5B promoter variant rs35705950 is a pathogenic determinant independent of other mutations remains to be elucidated.TOLLIP, a regulator of innate immune responses involved in modulating Toll-like, receptor signaling resides at the same genetic locus with theh et al.  showed t MUC5B gene regulatory region.As mentioned above, the increased methylation in MUC5B promoter region may directly affect MUC5B expression. Conversely, methylation will also affect the binding of some transcription factors to the MUC5B gene transcriptional start site, numerous several transcriptional factor binding sites have also been identified, including the motifs binding forkhead box A2 (FOXA2), activator protein 1 transcription factor (AP-1), Sp1, NF-κB, TTF-1, TGT3, CREB, STAT, c-Myc, and others [ MUC5B gene expression  [COL1A2) [ TGF-β [MMPs) [ MUC5B [r motifs  and can s [VEGF) , type I [COL1A2) , transfo [ TGF-β , matrix β [MMPs) , and MUC [ MUC5B . MUC5B transcription, the MUC5B promoter region in three intestinal cancer cell lines has been sequenced and analyzed by Van Seuningen and collaborators [ MUC5B promoter contains a high number of GC and CACCC boxes; those are the motifs binding Sp1. In human airway epithelial cells, Chang's group [ MUC5B via binding to cis-Sp1 sites in MUC5B promoter. The further research by Wu et al. [ MUC5B promoter, thereby promoting MUC5B expression. At present, although there is a limit data about the regulation of MUC5B by Sp1 in IPF, Kum et al. [ MUC5B expression in IPF. In order to identify the regulatory regions involved inborators . They re's group  used chru et al.  has showm et al.  have proNF-kB Mediated Activation of MUC5B. According to the study by Van Seuningen et al. [ MUC5B promoter are found to be nearby the binding motifs for other transcriptional factors. It indicates that the clustered transcriptional factors including Sp1 may act together to modulate transcription of MUC5B [κB also participates in the regulation of MUC5B transcription. MUC5B promoter region contains several putative NF-κB binding sites in murine middle ear epithelial cells, and there are two possible NF-κB cis-sites in the −556 bp MUC5B promoter region and one NF-κB cis-site in the −229 bp region [κB cis-sites in their 5' upstream flanking sequences in human lung epithelial cells [κB and induction of MUC5B in distal airway epithelium, however, the study [κB in murine middle ear epithelial cells. With regard to the upstream signaling molecules of NF-κB activation, the phosphorylation of P38 MAPK has been confirmed by quite a few studies [n et al. , Sp1 binof MUC5B . Among tp region . Studiesp region , can actal cells , 79. Althe study  has demo studies , 80–82. AP-1 Mediated Regulation of MUC5B. AP-1, a dimeric complex of related transcriptional factors containing members of the JUN, FOS, ATF, and MAF protein families, regulates a wide range of cellular processes including cell proliferation, apoptosis, migration, and differentiation. Previous studies suggest that there are intersections between the NF-κB pathway and AP-1 pathways, and many stimuli activating NF-κB will also activate AP-1 [κB, c-Jun, and c-Fos. Considering the association between AP-1 and MUC5B, Lee et al. [κB are believed to be associated with the overexpression of MUC5B and lung injury in IPF in our study. ate AP-1 , 84 whosate AP-1 . The actate AP-1  have proe et al.  have proe et al.  proved tCREB Mediated Regulation of MUC5B. cAMP-response element-binding protein (CREB) could recognize the specific DNA sequences 5'- TGACGTCA- 3', which is known as cAMP response element (CRE) in the transcription regulatory region of many genes [ cis-CRE sites  in the MUC5B promoter to upregulate MUC5B expression in human primary airway epithelial cells [ny genes , 90. Theny genes , proteinny genes , and p90ny genes  as well ny genes . The phoal cells . Previoual cells  indicateal cells .κB, and CREB and activated MAPKs signaling in the overexpression of MUC5B in pulmonary fibrosis. However, the regulation of MUC5B is considered a cell-specific process, and the special roles of the transcriptional factors in MUC5B expression in distal airway epithelium in IPF patients have not been characterized. It deserves further study, and we believe the greater understanding of MUC5B regulation by transcription factors may provide a new perspective on the treatment of IPF.The above studies indicate the important role of the transcriptional factors SP1, AP-1, NF-Although the role of inflammation in IPF is controversial, inflammatory mediators are indeed critical factors in the pathogenesis of IPF . Meanwhiβ, IL-4, IL-6, IL-9, IL-10, IL-13, IL-17, TNF-α, and IFN-γ, are known to regulate mucin synthesis [β, IL-6, IL-13, and IL-17A have been proved to upregulate MUC5B in airway epithelial cells in many studies [Over the last decades, considerable evidence has suggested that cytokines play important role of stimuli in pulmonary fibrosis development . Releaseynthesis , 107. Of studies –101.β is found to be upregulated [β in rat lungs promotes the presence of myofibroblasts, fibroblast foci, and extracellular matrix accumulation which are the characteristics of pulmonary fibrosis [β is also elevated in lungs of patients with MUC5B hypersecretion respiratory disease, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) [ MUC5B overexpression. Further study suggested the binding of IL-1β and IL-1R1 is followed by NF-κB-based transcriptional mechanism [β induced MUC5B overexpression in airway epithelium participates the development of IPF.In pulmonary fibrosis, IL-1egulated , and thefibrosis . IL-1β isis (CF) , 110–112sis (CF)  tested techanism . Therefoγδ+ T cells, has been confirmed to induce significant neutrophilia and pulmonary fibrosis after exposure to bleomycin [β dependent, suggesting the cooperative roles for IL-17A and TGF-β in the development of fibrosis [β are both increased in the bronchoalveolar lavage fluid of patients with IPF, and the fibrogenic effect by IL-1β is also dependent on IL-17A. Therefore, the increased IL-17A also plays a critical role in pulmonary fibrosis. Fujisawa et al. [β and IL-17A could active MUC5B promoter resides within the -4.17kb to -2.56kb region relative to MUC5B promoter transcriptional start site, containing putative NF-κB binding sites. The activated NF-κB based transcriptional mechanism by IL-17A is involved in MUC5B regulation in human bronchial epithelial cells [IL-17A, produced by CD4+ and leomycin –115. Blefibrosis . In addifibrosis  also shoa et al.  show thaal cells , 116. M. pneumoniae, viruses, or cigarette smoking will lead to the production of IL-13 which is a critical driver for mucus production [α, thus activated transcription 6 (STAT6) signaling pathway in airway epithelial cells [IL-13 typically serves a protective role and has been implicated as a key regulator of epithelial cell biology. However, disregulated IL-13 will exert powerful profibrogenic effects in numerous chronic fibrotic diseases, including interstitial lung disease , 118. Anoduction , 120. Pral cells , which ial cells , 122. Sial cells . MUC5B expression by IL-6 has been proved in human tracheobronchial epithelial cells in Chen's study [ MUC5B expression through the ERK signaling pathway.The cytokine IL-6 functions as a proinflammatory factor as well as a profibrotic factor in pulmonary fibrosis , 125, an's study , and the MUC5B in pulmonary fibrosis.Taken together, the studies indicate the important role of cytokines above and their related downstream signaling molecules in the overexpression of Staphylococcus enterotoxin are the most representative bacterial components that can induce MUC5B production.A number of bacterial components have been implicated in mucin gene regulation , 128. Li MUC5B expression in human NCI-H292 airway epithelial cells [ MUC5B by LPS induces TLR4 expression and the phosphorylation of ERK1/2 and p38 MAPK. Conversely, TLR4 siRNA-mediated knockdown of TLR4 significantly blocked LPS-induced MUC5B expression. These findings suggest that TLR4, ERK1/2 MAPK, and p38 MAPK play essential roles in LPS-induced MUC5B expression in human airway epithelial cells.LPS is located on the outer membrane of gram-negative bacteria and recognized by LPS-binding protein (LBP), CD14, and Toll-like receptor 4 (TLR4) leading to a robust proinflammatory response in mammalian cells including macrophages, epithelial cells, and fibroblasts . A lot oal cells , 131. Baal cells  have con Staphylococcus aureus enterotoxins are known to induce an inflammatory airway response. Although there is little information on the possible role of Staphylococcus aureus enterotoxins in the development of pulmonary fibrosis, however, previous animal study has demonstrated that the intratracheal administration of Staphylococcus aureus enterotoxins A and E could induce development of interstitial pneumonia [ Staphylococcus aureus enterotoxin B could induce endoplasmic reticulum stress via reactive oxygen species production in airway epithelial cells. Endoplasmic reticulum stress plays an important role in the regulation of MUC5B via the activations of X-box binding protein 1 (XBP-1), activating transcription factor 6 (ATF6), and CCAAT-enhancer-binding protein homologous protein (CHOP) in human airway epithelial cells [ Staphylococcus aureus enterotoxin A induced MUC5B expression by significantly inducing Toll-like receptor 2 (TLR2) expression and activating phosphorylation of ERK1/2 and p38 MAPK in human airway epithelial cells.Similar to LPS,neumonia . In the neumonia , they real cells . In addial cells  showed tROS generated from oxidative stress are implicated as important molecular mediators in the development of fibrosis in a variety of organs including the lungs. However, the causal role of ROS in mediating MUC5B overproduction has not been firmly established.κB signaling pathway, for example [ MUC5B expression via ROS formation and the NF-κB signaling pathway in human airway epithelial cells.ROS produced by cytokines, growth factors, and vasoactive agents contribute to the intracellular signaling cascades associated with inflammatory responses, including the ROS-dependent NF- example . A study example  showed v TERT and TERC [ MUC5B promoter region in some patients with sporadic IPF and familial pulmonary fibrosis, further studies are necessary to determine how ROS adversely impacts the MUC5B gene and its protein product in the development of pulmonary fibrosis in humans.Alternately, higher levels of ROS may trigger DNA damage, p53 activation, cell cycle blockade, and apoptosis. It is well known that the most common gene mutations identified in familial pulmonary fibrosis patients and some sporadic IPF patients are in the telomerase genesand TERC . These mand TERC , 138. Co MUC5B gene expression, Choi et al. [ MUC5B gene expression in airway epithelial cells. The study suggests that PGD2 acts as a key factor of MUC5B gene expression via the D-prostanoid receptor (DP1) in human airway epithelial cells. PGD2 binding to DP1 induced the ERK MAPK/RSK1/CREB activation. The p90 ribosomal S6 kinases (RSK) are a family of serine/threonine kinases that are activated downstream of the Ras/MAPK pathway. RSK phosphorylates multiple signaling effectors to play an essential role in numerous cellular functions, including regulation of gene expression through the phosphorylation of transcriptional regulators such as CREB. RSK1 is a mediator of CREB phosphorylation at Ser-133 and p-CREB strongly binds to the chromatin regions of CRE binding sites in the MUC5B promoter region, thereby inducing MUC5B gene expression in bronchial epithelial cells.Prostaglandin (PG) 2 is a potent biologically active lipid mediator that is produced from arachidonic acid by almost every type of cell . One of i et al.  investigInflammation is well known to be involved in the recurrent injury and repair in IPF. Therefore a better understanding of various inflammatory mediators that influence MUC5B overproduction in airway epithelial cells is potentially important for establishing a therapeutic strategy for treating MUC5B overproduction and IPF. MUC5B in the development of IPF and the potential mechanisms of MUC5B overexpression in airway epithelial cells. First, the localization of MUC5B in IPF lungs suggests a prominent role for injury and abnormal repair to the distal airway epithelium cells in IPF, inconsistent with the traditional view that IPF is a disease of the alveolar epithelium. It suggests that MUC5B overexpression in the distal airway may play a role in the development of IPF. Second, although there are limited data regarding the mechanisms of MUC5B overexpression in distal airway epithelium in IPF lungs, various studies have demonstrated that many factors modulate the expression of MUC5B. Besides the promoter variant rs35705950, which definitely causes an overexpression of MUC5B and appears to be predictive of IPF, some transcriptional factors, inflammatory mediators, and associated signaling pathways may also mediate MUC5B overexpression, providing clues for further research regarding the mechanisms of MUC5B overexpression in IPF, and may facilitate development of novel treatments for this fatal disease with limited treatment options.This review aimed to demonstrate the importance of


Despite that graphene has been extensively used in flexible wearable sensors, it remains an unmet need to fabricate a graphene-based sensor by a simple and low-cost method. Here, graphene nanoplatelets (GNPs) are prepared by thermal expansion method, and a sensor is fabricated by sealing of a graphene sheet with polyurethane (PU) medical film. Compared with other graphene-based sensors, it greatly simplifies the fabrication process and enables the effective measurement of signals. The resistance of graphene sheet changes linearly with the deformation of the graphene sensor, which lays a solid foundation for the detection of physiological signals. A signal processing circuit is developed to output the physiological signals in the form of electrical signals. The sensor was used to measure finger bending motion signals, respiration signals and pulse wave signals. All the results demonstrate that the graphene sensor fabricated by the simple and low-cost method is a promising platform for physiological signal measurement. Flexible and wearable sensors have drawn extensive attention due to their wide potential applications in wearable electronics. Flexible sensors with high sensitivity, good flexibility, and excellent stability are highly desirable for monitoring human biomedical signals, movements and environmental factors ,2. To endization ,11. Its strength 5 GPa [12125 GPa) ,14. Grap125 GPa) ,16,17. G125 GPa) . It is p125 GPa) ,20.A variety of graphene piezoresistive sensors have been developed for detecting human motions and spatially resolved monitoring of pressure distribution ,22,23. GIn this paper, a graphene sensor is demonstrated for physiological signal measurement. Graphene nanoplatelets (GNPs) are synthesized by thermal expansion method, and the graphene sensor is fabricated by double-sided sealing of polyurethane (PU) medical film on the graphene sheet. Three kinds of physiological signal are measured, including finger bending signals, respiratory signals and wrist pulse signals. A signal processing circuit composed of a signal conversion circuit and a filter circuit is developed to acquire and transmit the physiological signals. The GNPs synthesized by thermal expansion are cheap, conductive and already commercially available compared with CVD graphene and liquid phase exfoliated graphene . Due to The graphite intercalation compound was bought from Asbury Graphite Mills Inc . PU films were bought from Shanghai Hons Medical Technology Co. Ltd. . The tensile measuring of the GNP sheet was performed on a tensile measure machine TST-01 . Scanning Electron Microscope (SEM) images of the GNP sheets were obtained by JEOL JSM-7800FPRIME . The X-ray diffraction (XRD) spectra of graphite and GNPs were characterized by X-ray polycrystalline diffractometer APD 2000 PRO . The resistances were recorded by a Hydra Series III Data Acquisition System Fluke 2638A . The output voltages of the signal processing circuit were recorded by a data acquisition instrument NI USB-63619 .GNPs were prepared by thermal expansion and exfoliation method . The theAs shown in 2 of 0.98 at pressure ranging from 0 to 20 KPa and R2 of 0.99 at pressure ranging from 20 to 80 KPa. The changes of relative resistance variations of the GNP sheets have a bilinear relationship with the variation of the pressure applied to the sheet. The pressure response of the GNP sheets verifies that the GNP sheets have a high relative sensitivity at low pressures (0 to 20 KPa), which is suitable for practical applications . In addition, the piezoresistive properties of the GNP sheets are measured and analyzed. A GNP sheet connected with conductive wires was fixed at both ends on the tensile measuring machine. The GNP sheet was stretched slowly. In the meantime, its resistances were recorded at different stretching lengths. The experimental results were calculated using 3 repeated sheets. The initial resistance R0 of the GNP sheet is 6.5 Ω. 2 of 0.98. This fitting line shows that the change of relative resistance variation of the GNP sheet has a linear relationship with the variation of the stretching strain of the GNP sheet, which provides a good foundation for measuring physiological signals based on the graphene sensor. The GNP sheet is relatively flexible due to its condensed multiple-layer structure. It can bend to almost 270 degrees ,The measured resistance RThe linear range of the output voltage of the signal conversion circuit is between −2.4 V and 3.6 V due to the AD620 accessing a direct current (DC) power of 5 V. In the experiment of physiological signal measurements, the output of the bridge circuit conforms to the linear range when the graphene sensor is placed in the bridge circuit, which benefits the physiological signals measurements without signal distortion. 7, R8, R11, R12, C1, C2, C3 and C4 are parameters of the filter circuit and can be changed according to the required filter frequency range of the detection signal. The high cut-off frequency of the filter circuit can be expressed as in Equation (3),To improve the signal-to-noise ratio and reduce the noises produced by external noise and internal power frequency noise, filter circuits with different filtering ranges for measuring different signals are fabricated. As shown in The low cut-off frequency is expressed as in Equation (4),The filter circuit not only possesses a filtering function, but also an amplification function. The amplification factor can be expressed as in Equation (5),The signal conversion circuit converts the resistance signal acquired by the graphene sensor to a voltage signal. The filter circuit removes the noise from the signals. The whole system makes it possible to improve the signal-to-noise ratio and amplify the voltage amplitude of the signal by 11.4 times.Finger bending action measurement is of great importance in the field of human health and robotics . The preWe apply the graphene sensor to monitor finger bending signals in real time. We choose the low-pass filter circuit at 10 Hz to filter the finger bending signal, and collect the signal with a sampling frequency of 10,000 Hz using the data acquisition instrument (Video). Respiration is an important physiological process of the human body. Detection of respiration signals is an important part of healthcare monitoring. Diseases related respiration signals have attracted wide attention all over the world. The incidence of obstructive sleep apnea syndrome (OSAS) is high and harmful. Accurate and reliable monitoring of patients’ respiratory and sleep physiological signals can help to cure disease . At presPulse signal is an auxiliary examination of cardiovascular diseases. High pulse rate has a high correlation with the incidence of coronary heart diseases . It can The graphene sensor is applied to measure wrist pulse signals. We attach the graphene sensor to the wrist to measure the radial artery pulse wave. The pulse wave pressure on the wrist skin surface is less than 0.6 KPa ,38, whicIn short, we synthesized GNPs using the thermal expansion and exfoliation method, and developed a graphene sensor. The resistance of the graphene sensor has a linear relationship with its stretching strain, which lays a solid foundation for the measurement of physical signals. We fabricated a signal processing circuit to output the physiological signals collected by the graphene sensor. The data of the finger bending signal, respiration signal and wrist pulse signal were processed and analyzed, which confirmed the accuracy of the signals. The graphene sensor fabricated by this method provides a low-cost, simple and flexible sensing method for measurement of physical signals. This is believed to be promising in applications of rehabilitation training, sleep monitoring, and heart disease prevention.