The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are laterally segregated into functional domains as well as asymmetrically distributed between the outer and inner leaflets. Additionally, the spatial distribution and organization of these lipids dramatically change in response to various cellular states, such as cell division, differentiation, and apoptosis. Division of one cell into two daughter cells is one of the most fundamental requirements for the sustenance of growth in all living organisms. The successful completion of cytokinesis, the final stage of cell division, is critically dependent on the spatial distribution and organization of specific lipids. In this review, we discuss the properties of various lipid species associated with cytokinesis and the mechanisms involved in their polarization, including forward trafficking, endocytic recycling, local synthesis, and cortical flow models. The differences in lipid species requirements and distribution in mitotic vs. male meiotic cells will be discussed. We will concentrate on sphingolipids and phosphatidylinositols because their transbilayer organization and movement may be linked via the cytoskeleton and thus critically regulate various steps of cytokinesis. Division of one cell into two daughter cells is among the basic and essential functions of cells for their proliferation and growth. Animal cell membranes undergo extensive remodulation in preparation for cell division. At the onset of mitosis, cells shift their shape from being flat at interphase to round at metaphase and elongated ovoid shape at anaphase  1,2]. D,2. D1,2]A growing number of studies suggest that successful assembly of the actomyosin ring, cleavage furrow ingression and abscission at the midbody critically depends on lipid trafficking and polarization ,10. LipiBox 1Although cytokinetic failures are normal and important for certain physiological functions such as differentiation of hepatocytes, cardiomyocytes, vascular smooth muscle, megakaryocytes, and germ cell differentiation, cytokinetic failures in certain tissues induce pathological states such as cancer, certain blood diseases, female infertility, Lowe syndrome, neurofibromatosis type II, sphingolipidoses, and age-related macular degeneration . Here weSphingolipidoses:Sphingolipidoses are a group of inherited lysosomal storage disorders caused by deficiency of lysosomal enzymes, such as β-galactosylcerebrosidase in Krabbe disease, β-glucocerebrosidase in Gaucher disease, Globotriaosylceramidase in Fabry disease and acid sphingomyelinase in Niemann Pick type A&B ,15. PatiLowe syndrome:2 homeostasis on endosomal membranes and is required for cytokinesis  and one triphosphate, PIP3 [2 or FYVE-CENT binds to PI(3)P). PIPs are uniquely distributed in the subcellular membrane compartments. PI(4)P is localized to PM, endosomes, and the trans Golgi. PI(3)P is found in early endosomes, whereas PI(5)P is found on the PM, endosomes, nuclear envelope. PIP2 localized to PM, recycling endosomes, and lysosomes; PIP2 to PM and early endosomes; PIP2 to late endosomes and lysosomes; PIP3 is localized to the PM and endocytic compartments [2, PIP2, and PIP3 [Phosphatidylinositols (PI) are glycerophospholipids with a myo-inositol head group. PIs are initially synthesized on the membranes of the endoplasmic reticulum and subsequently transported to other cellular membranes. The cytosolically exposed hydrophilic myo-inositol head group on the target membranes gets modified by selective PI-kinases or PI-phosphatases to generate unique PIP domains in response to various cellular cues . Phospho3,4,5)P3 ,89. Impoartments . To date3,4,5)P3 ,88,89.2 is the most extensively studied lipid in animal cell cytokinesis, owing to its obvious enrichment in cytokinetic furrows in various cell types and organisms, as well as its broad implications in lipid–lipid and lipid–protein interactions and signaling [2 levels is associated with cytokinetic defects in several model organisms and mammalian cells [2 normally localizes uniformly to the inner leaflet of the plasma membrane in mammalian cells during prophase; however, during cytokinesis, PIP2 is specifically enriched at the inner leaflet of the cleavage furrow [2 accumulation at the furrow is not universal because its enrichment was not observed in Drosophila spermatocytes [Dictyostelium [2 is required for adhesion of plasma membrane to the contractile ring [2 is required for localization of RhoA, a small GTPase required for assembly of the contractile ring [2. Furthermore, PIP2 regulates cytokinesis via exocytosis, endocytosis, and endocytic recycling [2 and γ-tubulin and produces PIP2 from PI(4)P. The ESCRT II component VPS36 binds to PIP2 and recruits, ESCRT III component CHMP4B in an ALIX independent pathway to mediate abscission in eye lens cells [2 production, its turnover and clearance also influence cytokinesis [PI4,5)Pignaling ,93,94,95an cells ,97,98,99e furrow ,42. Howeostelium , suggestile ring ,102. Manile ring ,105,106,ile ring ,105, Aniile ring , Syndapiile ring ,110,111,ile ring  and subuile ring , bind toecycling ,124,125.okinesis ,92,99. S,5Pignaliokinesis ,128,129.3 localized to polar regions; in contrast, PTEN, a PIP3 phosphatase, localized to concentrates at the cleavage furrow [2 accumulate at the furrow, thus effectively enriching PIP2, and depleting PIP3 at the furrow [3 specific probe tGPH (GFP fused to pleckstrin homology domain of Grp1/Steppke under tubulin promoter) was enriched at the cleavage furrow in dividing Drosophila spermatocytes indicating PIP3 localization to this region. However, tGPH enrichment at the furrow was observed only in intact cysts, i.e., spermatocytes encased by cyst cell membrane, and acute loss of cyst cell membranes and concomitant reduction in PIP3 accumulation at the furrow did not prevent ingression of cleavage furrow during cytokinesis in spermatocytes [3 enrichment at the furrow is currently unknown and warrants future investigation. A study on plasma membrane expansion during cellularization in Drosophila embryos found that PI P3 inhibited PIP2-dependent actomyosin contractility, promoting actomyosin network disassembly [3 probe, Steppke, localized to actomyosin networks and reduced tissue tension by inhibiting the actomyosin activity at the adherens junction during dorsal closure in Drosophila embryos [2 and PIP3 are likely to play distinct and antagonistic roles in the regulation of actomyosin ring contractility during cytokinesis.In mitotically dividing cells, PI(3) kinase and its product PIPe furrow . Similare furrow ,130. Intatocytes . The sigassembly . The PI by RNAi or its inhibition with myriocin affected cell cycle progression but not vesicular trafficking or lipid raft formation [Sphingolipids are essential for cytokinesis, as disruption of sphingolipid biosynthesis either by genetic ablation or pharmacological methods induces cytokinetic defects ,135,136.ormation . Very loormation .o domains) on the outer leaflet of the PM [γ (phospholipase C) are enriched in Lo domains, and their activation via tyrosine phosphorylation is essential for cytokinesis [γ activation leading to Ca2+ release is essential for cytokinesis [Sphingolipids such as SM and gangliosides in combination with cholesterol were shown to form liquid ordered domains P2 enrichment at the furrow, it has been proposed that SM could directly or indirectly interact with PIP2 to limit its diffusion as well as locally activate PI(4)P 5-kinase for confined PIP2 synthesis [o domains, transbilayer coupling, signal transduction, and cytoskeletal reorganization.Sphingomyelin (SM), a major sphingolipid, is normally present uniformly on the outer leaflet of PM, and, during cytokinesis, it specifically gets enriched at the cleavage furrow. Interestingly, SM at the outer leaflet regulates PIPe furrow . Depletimulation . Cleavagynthesis . Proteinynthesis ,141,142.S. pombe, and their integrity is essential for proper cytokinesis [o domain-associated proteins flotillins were shown to form large, stable domains in the plasma membrane and accumulate at a higher density in the cleavage furrow of hematopoietic cells [Cholesterol was shown to accumulate at the cleavage furrow in sea urchin eggs , more spokinesis ,144. Optokinesis . In humaokinesis . Lo domaic cells . Cholestic cells . Caveolaic cells ,150,151.ic cells ,153,154.ic cells .Budding yeast quadruple mutants are1Δ, are2Δ, dga1Δ, and Iro1Δ, that cannot synthesize triacylglycerols show abnormal cytokinesis and septation defects . TAGs wi2 synthesis [Drosophila sphingosine-1-phosphate lyase gene (Sply) and sphingosine kinase 2 were shown to be important for normal muscle development and reproductive organ function [Phosphatidic acid (PA) is yet another phospholipid enriched in purified midbodies from HeLa cells, and RNAi knockdown of its metabolic enzyme ABHD5 resulted in cytokinetic defects . Howeverynthesis ,160,161.ynthesis . Inhibitynthesis . Phosphoynthesis ,164. Thefunction ,166, butPS is synthesized in the endoplasmic reticulum and trafficked via the Golgi to the plasma membrane, where it is mostly localized to the inner leaflet. In animal cells, PS can be flipped from the inner leaflet to the outer leaflet, where it acts as ‘eat me’ signal that promotes phagocytosis to engulf the cells. Apoptotic cells activate scramblases that quickly expose PS to the outer leaflet . RelativDrosophila member of the very long chain fatty acid synthase (Elovl) gene, Bond, was shown to be important for cytokinesis in spermatocytes [Drosophila desaturase gene family members exhibit a sex-specific expression pattern [Cytokinesis in somatic cells results in the complete physical separation of cytoplasm between the daughter cells. In contrast, cytokinesis in spermatocytes is incomplete, where all the sister cells are interconnected via cytoplasmic bridges . The preatocytes . Bond muatocytes . Lipids atocytes . Drosoph pattern . Sphingo pattern ,271. P-e pattern .ghi mutants display normal actomyosin ring assembly, but the ring fails to constrict to completion, leading to furrow regression and cytokinetic defects [Ghi affects the d16/d14 (sphingoid base acyl chain length) ratio, with a net shift towards increased accumulation of sphingolipids with a d16 sphingoid base, suggesting a crucial role for sphingoid base acyl chain length in male meiotic cytokinesis [Drosophila males had more unsaturated fatty acids in their sphingolipids compared to females [Drosophila, is essential for male fertility [Drosophila spermatocytes did not rescue cytokinetic defects [The small regulatory subunit of serine palmitoyltransferase (ssSPT), Ghiberti , which is part of the Serine Palmitoyltransferase (SPT) enzyme complex, has been shown to be required for male meiotic cytokinesis ,272. ghi defects . Loss ofertility ,274. We  defects . Live ce defects .Why do male germ cells have high amounts of unsaturated fatty acid-containing lipids, and why are they important in male meiotic cytokinesis? Although exact mechanisms are currently unknown, the answer could lie in their widely known roles in signaling and modulation of membrane biophysical properties. Unsaturated fatty acid-containing lipids introduce more disorder in the membranes; thereby, they make membranes more fluid, reduce membrane thickness, increase lipid packing defects, reduce membrane rigidity/make membrane more flexible, affect membrane protein conformations, and increase sensitivity to oxidation  275,276276. SphiA typical animal cell requires a total surface area increase of about 26% for successful division, and meiotic cells require about 60% due to two rounds of rapid successive divisions ,282,283.Most of the cultured animal cells entering mitosis dramatically change their shape from being flat at the interphase to round/spherical at the metaphase ,285,286.Caenorhabditis elegans embryos [Drosophila embryos [Drosophila spermatocytes undergoing meiotic divisions have demonstrated crucial roles for Golgi-derived vesicular trafficking in cytokinesis [2, a major phosphoinositide, by PI4P 5-kinase, which is found in the cytokinetic furrow and intercellular bridges and whose enzymatic activity is required for cytokinesis [Drosophila neuroblasts by promoting the synthesis of a plasma membrane-specific pool of PI4P by PI4KIIIα. Further, PI4P in turn was shown to bind to non-muscle myosin II regulatory light chain (Sqh) in vitro, suggesting a mechanism for anchoring of myosin to the cell cortex in neuroblasts [S. pombe revealed that scaffolding protein efr3-mediated PI4-kinase PM attachment is required for central positioning of contractile rings, without which contractile rings slide to one end in a myosin-V-dependent manner [Vesicle transport from the ER to the Golgi and then to the cleavage furrow was shown to be essential for cytokinesis in multiple animal model organisms, including Xenopus , Caenorh embryos , Drosoph embryos ,295, and embryos , and to  embryos ,297. Sevokinesis ,304,305.okinesis ,307,308.okinesis . Importaokinesis ,301,310.okinesis . Similarokinesis ,312. Wilokinesis . PI4P isokinesis ,95. PITProblasts . A studyt manner ,256. GOLt manner . GOLPH3 t manner .Relatively little is known about the role of other lipids in forward trafficking and cytokinesis. Very long chain sphingolipids have been shown in plant cells to be required for the fusion of Golgi-derived vesicles to the cell plate during cytokinesis . SphingoIt was once believed that endocytosis was inactive during cell division. However, subsequent studies demonstrated that endocytosis is active and has an important role during cytokinesis ,319,320.Drosophila cellularization in embryos revealed an important role for Rab11-positive recycling endosomes in cytokinesis [Drosophila and mammalian cells [2 levels at the intercellular bridge via direct interaction with PIP2 phosphatase (OCRL) to promote localized cortical actin remodeling required for intercellular bridge stability and abscission [Pioneering studies conducted on okinesis ,324 and okinesis . Severalokinesis ,232,325.okinesis ,327. It okinesis . siRNA san cells . Rab35 hscission ,7. Sagonscission . These escission . It was scission .Drosophila spermatocytes; instead, it localized to mitochondrial membranes [Recently, we have shown that the sphingolipid CPE is enriched in Rab7 (which marks late endosomes) and Rab11-positive endosomes that are specifically localized to the cleavage furrow during male meiosis cytokinesis. Further, localization of CPE-enriched endosomes to the cleavage furrow is dependent on the wild-type function of Rab11. The absence of CPE or interfering with the functions of Rab7, Rab11, or Rab35 significantly increases cytokinetic defects, suggesting that specific lipid traffic via the endocytic pathway is essential for cytokinesis. However, unlike Rab7 and Rab11 endosomes, Rab35 did not localize to the endosomal compartment in embranes , suggestDrosophila spermatocytes, fusion of MVEs with the cleavage furrow was not observed; instead, it was shown that MVEs release their intraluminal vesicles in the cytosol in the vicinity of the growing furrow, suggesting membrane delivery to the furrow membranes via small pockets P 5-kinase are found in the cleavage furrow and that overexpression of kinase-deficient PIP5K causes cytokinesis defects in CHO cells [S. pombe, and mutants of Its3 delayed cytokinesis [Drosophila cells, PI(4)P 5-kinase, Skittles, and PTEN, a PIP3 phosphatase, both produce PIP2 and localize to the cleavage furrow [2 in furrow membranes and the localization of PI4(P) 5-kinase suggest that PIP2 local synthesis is important during cytokinesis [2 has been linked to detergent-resistant membranes in several studies [2 accumulation in the inner leaflet is disrupted [2 production [2, and PI(4)P 5-kinase concentrate at the cleavage furrow. Anchored picket fence model, combined with a lipid raft and cortical flow mechanism involving the actin cytoskeleton, may explain this phenomenon, given that sphingolipids are not synthesized locally at the cleavage furrow Phin eggs ,350. Addhin eggs . A systehin eggs . PI 4-kiile ring . The corosophila . Psychososophila . Psychososophila . Thus, iEukaryotic cells invest a significant amount of genetic information and energy to produce and degrade thousands of lipid species, whose complexity and biological roles are only beginning to be explored. Lipid composition dramatically changes between each cell type and within the same cell type depending on the different cellular states in which they are examined, for instance when they are dividing or differentiating ,352. In Midbody lipidomes revealed a number of novel lipids, including ceramides, ether phospholipids, phosphatidic acid, and triacylglycerol; however, their roles in cytokinesis remain unknown . SphingoDrosophila have shown that very long chain unsaturated fatty acid containing sphingolipids are essential for male meiotic cytokinesis [Drosophila male meiotic cells, they fundamentally differ in the release of ILVs. Lysosomes release their ILVs outside the cell as exosomes when they fuse; in contrast, in our study, we find that MVBs release their ILVs in the cytosol proximal to ingressing membranes at the furrow [Long chain PUFAs as components of phospholipids and sphingolipids have been detected in the spermatozoa of most mammals, including humans and birds. Unsaturated fatty acid-containing lipids provide the spermatozoan plasma membrane with the fluidity and flexibility that it needs to participate in membrane fusion events during fertilization, although they make the plasma membrane more susceptible to oxidation . Studiesokinesis ,356. Ourokinesis . Howevere furrow ,329,330.e furrow ,356. HowC. elegans embryos warranted that, the cortical flows play only a minor role in actomyosin ring assembly and cytokinesis [There is overwhelming evidence showing polarization of specific lipids at the cytokinetic furrow, and intercellular bridges ,8. Howevokinesis . Severalokinesis . HoweverLastly, sphingolipids and cholesterol-rich domains are known to co-cluster several transmembrane receptor proteins that mediate signaling . Enrichm


Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy.Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry.+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs.Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC. Immune checkpoint inhibition (ICI) therapy is emerging as a promising new therapy for the treatment of advanced hepatocellular carcinoma (HCC).Only a minority of HCC patients will respond to ICI therapy and recent data suggest that HCC on the background of non-alcoholic steatohepatitis (NASH) may have reduced sensitivity to this treatment strategy.Neutrophils are a typical myeloid component of the liver in NASH and are found either within the HCC tumour microenvironment or in a peritumoural location.Neutrophils have considerable phenotypic plasticity and can exist in both tumour promoting and tumour suppressing states.Neutrophils may have the ability to influence ICI therapy.+ neutrophils are found in human NASH and within the tumour of both human and mouse models of NASH-HCC.CXCR2The resistance of NASH-HCC to anti-PD1 therapy is overcome by co-treatment with a CXCR2 small molecule inhibitor, with evidence of reduced tumour burden and extended survival.Anti-PD1 and CXCR2 inhibitor combine to selectively reprogramme tumour-associated neutrophils (TANs) from a protumour to an anti-tumour phenotype.+ immune clusters that contain physically associating CD8+ T cells and antigen presenting cells.Reprogrammed TANs proliferate locally within Granzyme B+ dendritic cells are found to be elevated in anti-PD1 and CXCR2 inhibitor treated HCCs and together with CD8+ T cells are required for therapeutic benefit.Conventional XCR1TANs can be selectively manipulated to adopt an anti-tumour phenotype which unlocks their potential for cancer therapy. The ability of CXCR2 antagonism to combine with ICI therapy to bring about enhanced therapeutic benefit in NASH-HCC  warrents clinical investigation.Primary liver cancer is emerging globally as one of the most common and deadly malignancies with 905 000 new diagnosed cases and 830 000 deaths recorded in 2020.+PD1+ T cells, that appear to promote, rather than suppress, NASH-HCC.Possible curative options for HCC such as tumour resection, liver transplant or ablation are at present limited to a minority of patients who are diagnosed at an early stage of the disease.+ T cells, B cells and CD11b+CD15+GranzymeB+ neutrophils. When considering the growing evidence for both pro-tumour and anti-tumour functions for neutrophils in a variety of cancersRecent use of imaging mass cytometry (IMC) and single cell sequencing to probe the cellular constituents of human HCC revealed considerable heterogeneity within the tumour microenvironment with intratumoural region-specific distributions of immune cells.+ T cells and conventional dendritic cells (cDC). Reshaping the tumour immune microenvironment was associated with a T cell- and DC-dependent reduction in tumour burden and increased survival. We propose that combined neutrophil phenotype modification and ICI may achieve improved outcomes in NASH-HCC.Here, we determined that the CXC chemokine receptor, CXCR2 is almost exclusively located on neutrophils in human and mouse NASH-HCC. This finding led us to ask if antagonism of CXCR2 can combine with anti-PD1 to overcome resistance of NASH-HCC to immunotherapy. Our findings suggest that this combination therapy reprogrammes the phenotype of tumour neutrophils and enhances their association with CD8+ and CD3+ immune cell infiltrates were increased in anti-PD1 treated ALIOS fed mice  and calcium  signalling  for the chemokine receptor CXCR2, the latter identified as being predominantly expressed by Ly6G+ neutrophils, were all increased in tumour tissue  T cells . These dTreatments using either or both AZD5069/anti-PD1 were then investigated for their ability to suppress tumour growth in the DEN-ALIOS model . Tumour + T cells were significantly increased in both anti-PD1 and AZD5069/anti-PD1 therapy groups, with only anti-PD1 monotherapy significantly affecting CD4+ T cells  antigen presenting cells (APCs) that were found in the regions of interest with six out of seven AZD5069/anti-PD1 treated tumours that were examined by IMC , CD8+ cell (T cell) and CD11c+ cell (DC and a subset of macrophages) dynamics within the tumours of DEN/ALIOS mice  was then performed to further interrogate Ly6GIOS mice . PCLS fr T cells . Althoug T cells . Neutropcontrols . These d10.1136/gutjnl-2021-326259.supp4Supplementary videoet alJund, Csf3r, Rps27) . Lactoferrin (Ltf) was of particular interest among the early neutrotime genes as its protein has well documented anti-cancer activities; including the activation of DCs and macrophages and enhancing the cytotoxic properties of natural killer cells.Given that our observations were consistent with intratumoural granulopoiesis in response to combination AZD5069/anti-PD1 therapy, we more closely characterised the TAN phenotype under these conditions. Grieshaber-Bouyer , Rps27) . HoweverInterrogation of transcriptome data from NASH-related and non-NASH-related HCC patients identified the late neutrotime signature to be significantly enriched in NASH-HCC when compared with HCCs of other aetiologies+ neutrophils revealed that TANs from AZD5069/anti-PD1 treatment mice were enriched for process networks associated with cell cycle, phagocytosis and antigen presentation when compared with vehicle controls  we noted more pronounced suppressive effects when compared with the AZD5069 montherapy alone (Int neutrophil population isolated from lipopolysaccharide-(LPS)-treated miceRNA-Seq of purified Ly6Gcontrols . AZD5069controls . Anti-PDcontrols . These fpy alone . Hence, py alone . FinallyLo neutrophils in the blood and resulted in a significant reduction in tumour burden  of intratumoural XCR1+ cDC1 cells and elevated CD8+ T cells in tumours, unlike mice transfused with equal numbers of mature neutrophils  and monocytic Ly6G-Ly6CHi (M-MDSC) cells. Accumulating evidence suggests that PMN-MDSC are immunosuppressive neutrophils and may be functionally very similar to the TANs that have been termed ‘N2’, with shared protumour properties.+ T cell functions associated with the activities of PMN-MDSCs and immunosuppressive neutrophils were clearly overcome by combined AZD5069/anti-PD1 therapy.Neutrophil infiltration is a key pathological feature of human NASH that may result from upregulation of hepatic CXCL8 (IL-8) and CXCL1,+ cells and that is due to survival and expansion of residual tissue neutrophils driven by high systemic levels of granulocyte colony-stimulating factor,+ T cell counts and induction of anti-tumoural Gzmb; these being changes that were also noted with AZD506/anti-PD1 therapy. In future work it will be important to identify selective markers of the reprogrammed TANs that might be exploited for detailed functional characterisation, as well as for enabling their selective experimental manipulation which at present is not possible. Also, it will be important to determine precisely how and why combined CXCR2 antagonism and anti-PD1 treatment selectively induces proliferative immature neutrophils in the tumour. Clinically the ability to selectively reprogramme TANs while retaining mature anti-microbial neutrophils in the circulation may be very relevant in HCC since bacterial infections and septic shock are common clinical challenges in cirrhotic patients (in whom 90% of HCC develops).A growing body of evidence suggests that CXCR2 inhibition may be therapeutically beneficial in many human cancers including; pancreatic, lung, ovarian, prostate, colon and now the liver.In summary, we present a novel combination immunotherapy that enhances the efficacy of anti-PD1 in NASH-HCC. As the CXCR2 antagonist AZD5069 has been demonstrated to be safe for use in humans it is timely to determine if HCC patients would benefit from a similar therapy.https://www.nc3rs.org.uk/arrive-guidelines), and in accordance with the UK Animal (Scientific Procedures) Act 1986, and were subject to review by the animal welfare and ethical review board of the University of Glasgow and Newcastle University. All mice were housed in specific pathogen free conditions with unrestricted access to food and water and maintained on a constant 12 hours light-dark cycle under controlled climate . All animal experiments using the CD-HFD were performed in accordance with German law and the governmental bodies, and with approval from the Regierungspräsidium Karlsruhe . Male mice were housed at the German Cancer Research Centre (DKFZ) , adhered to ARRIVE guidelines  and R (V.3.5.1) performing tests as indicated and were considered statistically significant. P values are included in figures.Additional methods are described in 10.1136/gutjnl-2021-326259.supp2Supplementary data10.1136/gutjnl-2021-326259.supp3Supplementary data


Little is known about the role of the World Health Organization (WHO) in communicating with the public on social media during a global health emergency. More specifically, there is no study about the relationship between the agendas of the WHO and Twitter users during the COVID-19 pandemic.This study utilizes the network agenda-setting model to investigate the mutual relationship between the agenda of the WHO’s official Twitter account and the agenda of 7.5 million of its Twitter followers regarding COVID-19.Content analysis was applied to 7090 tweets posted by the WHO on Twitter from January 1, 2020, to July 31, 2020, to identify the topics of tweets. The quadratic assignment procedure (QAP) was used to investigate the relationship between the WHO agenda network and the agenda network of the 6 Twitter user categories, including “health care professionals,” “academics,” “politicians,” “print and electronic media,” “legal professionals,” and the “private sector.” Additionally, 98 Granger causality statistical tests were performed to determine which topic in the WHO agenda had an effect on the corresponding topic in each Twitter user category and vice versa.Content analysis revealed 7 topics that reflect the WHO agenda related to the COVID-19 pandemic, including “prevention,” “solidarity,” “charity,” “teamwork,” “ill-effect,” “surveillance,” and “credibility.” Results of the QAP showed significant and strong correlations between the WHO agenda network and the agenda network of each Twitter user category. These results provide evidence that WHO had an overall effect on different types of Twitter users on the identified topics. For instance, the Granger causality tests indicated that the WHO tweets influenced politicians and print and electronic media about “surveillance.” The WHO tweets also influenced academics and the private sector about “credibility” and print and electronic media about “ill-effect.” Additionally, Twitter users affected some topics in the WHO. For instance, WHO followers affected “charity” and “prevention” in the WHO.This paper extends theorizing on agenda setting by providing empirical evidence that agenda-setting effects vary by topic and types of Twitter users. Although prior studies showed that network agenda setting is a “one-way” model, the novel findings of this research confirm a “2-way” or “multiway” effect of agenda setting on social media due to the interactions between the content creators and audiences. The WHO can determine which topics should be promoted on social media during different phases of a pandemic and collaborate with other public health gatekeepers to collectively make them salient in the public. Social media has changed how online users share and receive news and information on various public health emergencies. A contagious and fatal public health emergency that started in early 2020 was the COVID-19 pandemic, with 263,563,622 confirmed cases, including 5,232,562 deaths worldwide as of December 03, 2021 )Past studies have suggested that agenda setting can have different effects on various public subgroups ,19,29. TThis study fills in these gaps in the literature, by studying the network agenda-setting effect of the WHO on 6 Twitter user categories (that follow WHO's Twitter account).The WHO’s followers on Twitter were classified into 6 categories, including (1) health care professionals, (2) academics, (3) politicians, (4) print and electronic media, (5) legal professionals, and (6) the private sector. This classification was done to investigate if and how different categories on social media react to a public health issue.We adopted and modified the approach used by Toupin et al  to classIn this study, health care professionals include public health workers in academia and industry sectors, such as doctors and nurses. Health care professionals serve citizens during public health emergencies in various ways, for instance, by warning them against self-medicating .Academics were defined as the people who work in academia , such as professors, researchers, and students who can inform society through their research and writing about various issues related to the COVID-19 pandemic .Politicians include policy and decision makers in the state and federal governments, such as mayors, congressmen, congresswomen, and senators. Politicians can impact people by, for instance, communicating with them about the concerns raised around vaccine safety .Print and electronic media include those responsible for information dissemination and public awareness, such as journalists, press, news agencies, and publishers. The print and electronic media are powerful sources of information for the public as a crisis unfolds . They caLegal professionals include courts, lawyers, and attorneys who provide legal advice and resources, for instance, for an ideal crisis communication strategy . AdditioPrivate sectors include corporations, incorporated organizations, companies, chief executive officers, and for-profits who serve society by satisfying the needs of citizens. For example, companies can take necessary actions to serve their customers by empowering their employees to operate remotely .The classification of followers into 6 categories was done according to the short biographical profiles of Twitter users. The data sets used in this study can be found online . R softwFirst, the IDs of the approximately 7.5 million Twitter accounts following the WHO in 2020 were retrieved, and the “username” associated with each ID was collected.Next, the “description” used in biographies of the accounts  were collected. Table S12 in To make each category as exclusive as possible and increase the reliability of the classification, general keywords, such as “research,” “researching,” “teams,” “organizations,” “institutes,” “campus,” “professional,” “officer,” “change,” “equity,” and “policy,” were excluded in classifying Twitter users. The preliminary investigation and analysis of user biographies indicated that some keywords such as “chief” or “boss” appeared in several categories. Such keywords were also excluded.brandwach platform, the current study collected 7090 tweets posted by the WHO from January 1, 2020, to July 31, 2020.Twitter was the main source for data collection. Twitter contains features such as hashtags, retweets, replies, mentions, and likes that make it a suitable platform for studying online social interactions . Using trtweet package.The tweets related to COVID-19 posted by each Twitter user category from January 1, 2020, to July 31, 2020, were collected using the tweetbotornot package in the R software to classify Twitter accounts into bots and nonbots; the package is 91.78% accurate in identifying bots and 92.61% accurate in identifying nonbots )Granger causality has been used in previous agenda-setting studies to examine the relationship between media agenda and public agenda eg, -53). Gra. Gra53])P value of a Granger causality test is less than .05, the independent variable is said to Granger cause or predict the value of the dependent variable [In this study, Granger causality was used, for instance, to examine if “credibility” as a topic that was promoted by the WHO on Twitter predicted the future values of “credibility” in the tweets posted by the WHO’s followers on Twitter. If the variable . For insTo investigate if each time series in the WHO  could predict the value of its corresponding time series  in any of the Twitter user categories, a vector autoregression (VAR) model was created. Additionally, to investigate if each time series in any of the Twitter user categories  could predict the value of its corresponding time series  in the WHO, VAR models were created. Overall, 98 VAR models were created in this study.To perform Granger causality, first, each variable  should be treated as a time series . This stTo create time series for each topic, the frequency was calculated over time from January 1, 2020, to July 31, 2020, for both the WHO and the 6 categories of WHO followers on Twitter. For each topic, a time series with 182 records was created. Each record contained the frequency of the topic on a specific date. The initial analysis indicated that each time series had many zero number values, which skewed the data. Each zero number represents a lack of data for a given topic on a given date. The highly skewed data violated the normality assumption .Additionally, having a wide range of values from zero to several hundred led to violating the heteroscedasticity assumption  in many cases. This study tried to resolve the normality and heteroscedasticity issues by aggregating the data to weekly data to reduce zeros. Therefore, for each topic, a time series of 26 weeks was created. Although the data were aggregated to weekly data, the first 2 weeks for most topics still included zero numbers, which again violated the normality assumption in some cases. Removing the first 2 weeks of June 2020 from the analysis resolved the normality issue. Therefore, the analysis was carried out on times series with 24 records . As an example, the times series for the topics of WHO followers  are presented in Table S10 in To perform Granger causality, VAR models should be created first, as explained below.t-1 and TWt-2 are the first and second lags of TW (first variable) and TU t-1 + TO t-2 are the first and second lags of TU (second variable). Each lag is x period ago. For instance, lag one is 1 time period ago or lag two is 2 time periods ago:A VAR model is used to determine how 2 or more times series influence each other. In a VAR model, each time series is modeled as a linear combination of past values of itself and the past values of other variables . For insIn a VAR model, a different number of lags can be considered for each variable. Thus, the optimal number of lags should be selected using a criterion. This study used the Akaike information criterion (AIC) to select the optimal lag for each VAR model, as explained in the following sections.The most commonly used criterion in lag selection in VAR models is the AIC ,57. The A major assumption underlying the VAR model and Granger causality is that time series must be stationary ; otherwiP value of the Augmented Dickey-Fuller test is less than .05. In this study, all variables in the WHO and the 6 Twitter user categories were nonstationary, which were transformed to become stationary through differencing (first or higher differences of the variables). Differencing is the process in which the differences between consecutive observations of a variable  are computed [A time series is stationary if the computed .P value in this test is less than .05, autocorrelation exists.A VAR model should be tested to determine if it “provides an adequate description of the data...In time series models, autocorrelation of the residual values is used to determine the goodness of fit of the model. Autocorrelation of the residuals indicates that there is information that has not been accounted for in the model” . The PorP value is larger than .05, the residuals of VAR model are normal. In 4 cases, BoxCox transformation was used to make the VAR model normal  test proposed by Engle  was usedThe first study objective was to identify the topics discussed by the WHO on Twitter about COVID-19. Using content analysis, 7 topics were identified inductively, including prevention (n=2430), solidarity (n=717), teamwork (n=601), surveillance (n=276), charity (n=243), ill-effect (n=213), and credibility (n=156). The numbers in the parentheses represent the frequency of each topic.“Prevention” refers to the tweets posted by the WHO about how to avoid contracting the virus, including content about disinfecting surfaces, hand washing, wearing masks, vaccination, social distancing, isolation, and staying home. “Solidarity” refers to the tweets emphasizing the importance of unity, resilience, kindness, or supporting groups like refugees. “Teamwork” was another topic discussed by the WHO to highlight that countries, governments, organizations, and people should collaborate, coordinate, cooperate, be committed, and be accountable to control the pandemic. “Surveillance” emphasizes that governments should keep tracing, investigating, monitoring, and screening people and regions affected by the virus to take necessary actions. “Charity” refers to the donations, fundraisings, and financial support received by the WHO from governments, organizations, companies, celebrities, or other countries to fight against the virus. “Ill-effect” refers to the WHO's tweets about the consequences of the virus, such as disruptions in the economy, trades, health systems, mental health, abuse, and home violence during the quarantine. “Credibility” includes the tweets that demonstrate the importance of facts and bring people’s attention to the rumors, misinformation, and fake information related to COVID-19.This study also calculated the frequency of these topics for the 6 Twitter user categories and all of them together . In the data set of WHO followers, prevention  had the highest frequency, followed by solidarity , charity , teamwork , ill-effect , surveillance , and credibility . Table S13 in There was a strong tie between “prevention” and “solidarity” in in the matrix of the WHO and WHO followers and all 6 Twitter user categories. The strong tie between “solidarity” and “prevention” could indicate that the former is vital for the prevention of COVID-19 (or even treatment and response to the virus). For instance, to help prevent the spread of the virus, people should be united in following public health guidelines such as staying home, hand washing, and social distancing. These connections could represent social realities constructed around COVID-19 on Twitter and be transferred to the public agenda through network agenda setting .The QAP was used to measure the similarity between agenda matrices (networks). The QAP calculates the Pearson’s correlation coefficient between the 2 matrices. The co-occurrence matrices for the WHO followers and WHO are presented in Degree centrality is an important concept in network analysis, which indicates how important a node within the network is and will be calculated by the total number of connections a node has . In thisResults of the QAP tests indicated a positive and high correlation between the WHO’s agenda network and the agenda network of WHO followers see . The QAPQAP linear regression was also carried out to assess whether the WHO agenda network (the independent variable) could predict the agenda networks of Twitter user categories (the dependent variables). As evident in By performing 98 Granger causality tests, this study examined if the 7 topics in the WHO agenda Granger caused or predicted the future values of the topics in the tweets by the 6 types of Twitter users or vice versa. For instance, we tested to see if “teamwork” in the WHO Granger caused  “teamwork” in health care professionals. We also investigated if “teamwork” in health care professionals Granger caused “teamwork” in the WHO. This study uses the term “influenced” or “affected” instead of “Granger caused” or “predicted the value of.”F5,10=4.74, P=.01). On the other hand, among the 7 topics in the WHO followers, “charity” influenced “charity” in the WHO , and “prevention” influenced “prevention” in WHO .F6,6=5.13, P=.03) and “surveillance” in print and electronic media . Additionally, the WHO influenced “ill-effect” in print and electronic media , “credibility” in the private sector , and “credibility” in academics .The results indicated that the WHO influenced “surveillance” in politicians , “credibility” in politicians , and “credibility” in the private sector influenced “credibility” in the WHO . “Charity” in health care professionals  and “charity” in academics influenced “charity” in the WHO . “Prevention” in politicians  and “prevention” in print and electronic media  influenced “prevention” in the WHO. Print and electronic media influenced “surveillance”  in the WHO. The private sector also influenced “ill-effect” in the WHO . The WHO and legal professionals did not influence each other in any of the 7 topics.Twitter user categories also influenced several topics in the WHO’s agenda: “credibility” in academics . For instance, the WHO only influenced “ill-effect” in print and electronic media. Likewise, different Twitter user categories only influenced some  topics in the WHO agenda.It is hard to say who is leading the overall trends and topics related to COVID-19 on Twitter, mainly because social media provides unlimited space where various sources can interact with and impact each other . HoweverThis study was not designed to measure the impact of the WHO on top influencers within each type of Twitter user or vice versa. Future research can explore who sets the agenda and who establishes the agenda-setting effect first. It is possible that the WHO originates the tweets about COVID-19, but the top influencers within each Twitter user category set the agenda or promote the agenda already set by the WHO, through retweeting the WHO’s tweets.This study informs the network agenda-setting model by demonstrating that there can be a “2-way” relationship between the agenda of the WHO and its followers on Twitter. Vargo and Guo  also staThus, the WHO does not seem to set the public agenda in a unidirectional nature. Neither the WHO nor Twitter user categories play a leading role on social media because they influence each other’s agenda. It is possible that the WHO and different types of Twitter users pay attention to each other’s agenda and interact with each other on some topics related to COVID-19 in a bidirectional way .Vargo and Guo  also sugA limitation in most network agenda-setting studies is that it is not clear if the public agenda is directly impacted by gatekeepers  or if other sources are involved too in influencing public agenda. For instance, the study by Vu et al  comparedTo fill this gap, this study investigated 6 Twitter user categories that follow the WHO Twitter account. From the results of this study, it can be concluded that there are 2 levels of the agenda-setting effect on Twitter—one at the aggregate level (see ), that iThe results of this study can inform policy and be used to prepare for future pandemics in several ways: First, the WHO should define a clear strategy as how to use social media during pandemics to convey its messages to the public. The WHO should have a plan for what topics are more critical for the public during similar public health emergencies and work on transferring them to the public agenda.Second, how messages are framed and presented to the public is important. The WHO can identify the needs of social media users and provide information-related benefits for them by framing and presenting more effective messages during public health emergencies. The strategic framing of public health messages can help the WHO to have greater impact on social media users. For instance, to design more effective messages and attract more audiences, the WHO can frame its messages by using hashtags that are popular among the public .Third, using hashtags can lead to establishing and promoting important public health, social, political, or environmental agendas . HashtagFourth, this study suggests that there could be a “2-way” or “multiway” agenda-setting effect on social media. For instance, the WHO, the CDC, the US government, and politicians could all interact with and influence each other’s agenda. Therefore, in future public health emergencies, the WHO can determine which topics should be promoted on social media during different phases of a pandemic and collaborate with other gatekeepers such as the CDC to collectively make them salient in the public.This study investigated only 6 Twitter user categories. There are other types of users following the WHO on Twitter, which can be studied in future work, such as artists and athletes.Another limitation of using Twitter data is that Twitter users are not representative of the entire population.The agenda of WHO followers about COVID-19 could have been influenced by other resources too, such as the CDC or any other government agency. Future studies should find ways to also take the effect sources other than the WHO into consideration. Surveys and interviews with WHO followers on Twitter can provide more insights into the WHO's impact on social media users’ opinions about COVID-19.The 7 topics promoted by the WHO on Twitter could possibly also be found in the messages shared on Twitter by other resources such as the US government. It could be the case that the 6 categories of WHO followers on Twitter were also propagating the agenda by other resources and not specifically that of the WHO. Therefore, although this study suggests that there can be a “2-way” agenda-setting effect on social media, there could be a “multiway agenda-setting effect” on social media.This study was limited to tweets written in English. Analyzing tweets in other languages may provide new insights on the agenda-setting effect of the WHO on Twitter.Although this study analyzed a sample of tweets in the first 6 months in 2020, it is possible that the analysis of all the tweets posted by the WHO and its followers in the second half of 2020 could lead to different findings.We attempted to make the Twitter user categories as exclusive as possible; however, there might be some overlap among different categories. For instance, a Twitter user account could fall into the academics and print and electronic media categories.Future studies in this area can also determine to which social media user categories and top influencers within those categories the WHO should reach out if it wants to have more impact on social media users.Future studies in this area can also study whether and to what extent the WHO has made strategic use of hashtags to communicate its messages about COVID-19 to the public. Future research can also investigate the role of hashtags used by the WHO in setting its COVID-19 agenda and the effect of that agenda on public opinion.Although using social media data provides a legitimate method for studying the agenda-setting effect of WHO, more empirical research, including surveys and interviews, can be used in future studies to understand the role of the WHO in shaping public agenda on social media.The results show that the WHO and Twitter user categories of different types could influence some of the COVID-19–related topics in each other; however, this study did not investigate whether different Twitter user categories would also impact each other’s agenda network. This impact can be examined in future studies by exploring, for instance, the network agenda-setting effect between academics and health care professionals on Twitter.This is among the first studies that demonstrate the presence of network agenda-setting effects between the WHO and its followers on Twitter, specifically different Twitter user categories. In line with the network agenda-setting model, this study showed that the topics promoted by the WHO about COVID-19, such as “credibility” or “surveillance,” were linked together in a network.This study extends theorizing on agenda setting by providing evidence that agenda–setting effects vary by different Twitter user categories and topics. For instance, the WHO only influenced “surveillance” in politics and print and electronic media, or health care professionals only influenced “charity” in the WHO, while the WHO and legal professionals did influence each other.This study also extends theorizing on agenda setting by indicating that, while network agenda setting is known as a “1-way” model, there can be “2-way” or “multiway” effects of agenda setting on social media, because the influences between the WHO and Twitter user categories were reciprocal on Twitter.


Acute intraoperative fluid misdirection is a serious complication that may occur during phacoemulsification. We provide a detailed description and a video of our preferred technique for prompt management of acute intraoperative fluid misdirection.A 79-year-old male developed sudden shallowing of the anterior chamber and marked elevation of intraocular pressure at hydrodissection during phacoemulsification surgery. Treatment consisted of a needle aspiration of trapped fluid from the retrolental space, employing a 5/8th inch, 25-gauge needle on a medium-size syringe leading to immediate softening of the globe and deepening of the anterior chamber. The rest of the case proceeded uneventfully. The patient had uncomplicated recovery and final best-corrected visual acuity of 20/20.Acute subcapsular infusion fluid entrapment may occur during uneventful phacoemulsification. Needle aspiration of retrolental fluid is a simple and inexpensive method for immediate resolution of high IOP and deepening of the anterior chamber, allowing the case to proceed. Preoperative best-corrected visual acuity was 20/100. Topical anesthesia was applied. Trypan blue and Malyugin ring were utilized. During hydrodissection the anterior chamber was noted to suddenly shallow, and the eye became rock-hard. Efforts to express viscoelastic through the corneal wounds failed to soften the eye, and the iris was seen protruding into the main wound. Thus, a small amount of subconjunctival lidocaine was administered for patient comfort, and then a 5/8th inch 25-gauge needle on a 5-mL syringe was inserted through the conjunctiva at a distance of 4.0 mm posterior to the corneal limbus . The neehttps://doi.org/10.1016/j.ajoc.2022.101773.Supplementary video related to this article can be found at Video demonstrates our needle aspiration technique for management of acute intraoperative fluid misdirection. Subconjunctival lidocaine, needle insertion, initial “dry tap”, needle reposition, and fluid aspiration are demonstrated.Supplemental Video 1The following is the supplementary data related to this article:3The optimal treatment for acute irrigation fluid misdirection syndrome has been controversial. Liberation of retrolenticular fluid via a needle aspiration, one-port vitrectomy with utilization of a single small-gauge  trocar/cannula for vitrector insertion, and even a full three-port vitrectomy have all been described.Over the past 17 years in practice, we encountered approximately 6 cases of acute intraoperative fluid misdirection and successfully managed all by using a short 5/8th inch or 1-inch 25-gauge needle as described here. Since the iris-lens diaphragm is displaced anteriorly in these cases, and the needle enters the eye perpendicularly at 4.0 mm (if still phakic) or 3.5 mm (if pseudophakic) posterior to the limbus, the likelihood of injuring the lens or the posterior capsule is minimized. The technique is similar to a vitreous tap used for obtaining vitreous cultures. Typically, balanced salt solution will be seen to enter the syringe immediately or after repositioning the needle as shown in the video, and the eye will immediately soften. The rest of the case typically proceeds in a routine fashion without reaccumulation of misdirected fluid.We highly prefer a simple needle aspiration to utilizing a vitrector in these cases for several reasons. Firstly, the IOP can be immediately normalized since no additional instrumentation or machine set up is required, minimizing retinal and optic nerve ischemia in the setting of prolonged high IOP. Secondly, a needle aspiration carries a much smaller risk of inadvertently cutting the posterior capsule compared to a cutter. Additionally, a needle exerts far less traction on the vitreous base than a transconjunctival cannula and vitreous cutter. Since a retinal examination with scleral indentation may be difficult with a hazy cornea at the end of phacoemulsification, potential retinal breaks caused by the cannula insertion and the vitreous cutter, which are not uncommonly seen in vitreoretinal procedures, could result in a higher risk of retinal detachment. Finally, avoiding the need for opening additional instrumentation, seeking the assistance of a retinal surgeon to help with the vitrectomy, and avoiding unnecessary removal of the vitreous makes the procedure safer and saves time and cost to the facility. Alternatively, intravenous mannitol infusion can be given to dehydrate the vitreous humor by drawing out water which decreases vitreous mass and pressure. Unfortunately, this process is much slower than the direct fluid aspiration and thus less efficient.Positive pressure in the vitreous cavity can also develop when large amounts of viscoelastic are placed in the sulcus. When placing a sulcus IOL or when performing endocyclophotocoagulation in glaucoma patients, a cohesive viscoelastic material is typically injected into the sulcus space. However, viscoelastic can track posteriorly through the zonules, leading to positive pressure in the posterior chamber. This shift leads to shallowing of the sulcus space, which can mistakenly lead the surgeon to inject additional viscoelastic into the sulcus space, further exacerbating the positive pressure. A 25-gauge or a 27-gauge needle on an open 3-mL syringe can be inserted 3.5mm posterior to the limbus in a pseudophakic eye through the pars plana. Left open to air for 10–15 seconds or with application of active aspiration, this syringe will allow for the egress of excess viscoelastic, thereby relieving the positive pressure in the posterior chamber.4In summary, aspiration of retrolental fluid with a short 25-gauge needle on a medium-size syringe is a simple and inexpensive method for immediate resolution of high IOP and shallow anterior chamber in cases of acute intraoperative fluid misdirection.Written consent to publish this case has not been obtained. This report does not contain any personal identifying information.No financial support was received for the work being submitted, and none of the conflicts of interest listed are relevant to this work.All authors attest that they meet the current ICMJE criteria for Authorship.NZG reports grant support from Biogen and Applied Genetic Technologies Corporation, consultation honoraria from Bionic Vision Technologies, and travel expenses from Medical Conference Planners International. SS reports consultant fees from Sight Sciences, Ivantis, Lumata Health, and Abbvie as well as equity in Lumata Health and sponsorship from Heidelberg Engineering. The following authors have no finandical disclosures: PS and RZ.


Hypoxia in solid tumors is associated with poor prognosis, increased aggressiveness, and strong resistance to therapeutics, making accurate monitoring of hypoxia important. Several imaging modalities have been used to study hypoxia, but each modality has inherent limitations. The use of a second modality can compensate for the limitations and validate the results of any single imaging modality. In this review, we describe dual-mode imaging systems for the detection of hypoxia that have been reported since the start of the 21st century. First, we provide a brief overview of the hallmarks of hypoxia used for imaging and the imaging modalities used to detect hypoxia, including optical imaging, ultrasound imaging, photoacoustic imaging, single-photon emission tomography, X-ray computed tomography, positron emission tomography, Cerenkov radiation energy transfer imaging, magnetic resonance imaging, electron paramagnetic resonance imaging, magnetic particle imaging, and surface-enhanced Raman spectroscopy, and mass spectrometric imaging. These overviews are followed by examples of hypoxia-relevant imaging using a mixture of probes for complementary single-mode imaging techniques. Then, we describe dual-mode molecular switches that are responsive in multiple imaging modalities to at least one hypoxia-induced pathological change. Finally, we offer future perspectives toward dual-mode imaging of hypoxia and hypoxia-induced pathophysiological changes in tumor microenvironments. MPI use15-fold ..2 for MRT1-weighted contrast in MRI and fluorescence intensity under hypoxia-induced stimuli, including low pH and a reducing environment  that undergoes color changes and shifts in the SERS spectrum carbonyl stretching vibration in the presence of thiol  and selectivity for H2S over other thiols. Cleavage of the C–N bond of 26a by H2S and formation of 26b results in a bathochromic shift and decrease in fluorescence signal. On the other hand, due to the strong absorption of light by 26b, the photoacoustic signal is enhanced. Therefore, the probe exhibits fluorescence quenching and photoacoustic signal enhancement in response to the presence of H2S. Studies with HeLa cells treated with 26a demonstrated a decrease in fluorescence signals in the presence of H2S (200 μM) and a strong photoacoustic signal that is about 2.9-fold larger than that of the control group consisting of only 26a without H2S . C. C2S-spe27a and 27b, were induced to self-assemble with human serum albumin to form albumin–dye nanocomplexes. Absorbance and fluorescence of 27b are inert to pH changes, and thus 24b serves as an internal reference. Dye 27a is pH-responsive and acts as a pH indicator under both ratiometric photoacoustic and fluorescence imaging. In vivo applications of the nanoprobe as a pH indicator were demonstrated using tumor-bearing mice  nanoparticles that detect endogenous H2S in colon tumors [2S in the cellular environment can partially vulcanize Cu2O in the shell to form porous copper sulfide and, consequently, enhance near-IR absorption resulting in photoacoustic signals. Moreover, due to the formation of the holes, inner-sphere water exchange at the Fe3O4 core is accelerated, generating contrast enhancement in T1-weighted MR images. In vivo studies performed using tumor-bearing mice with elevated levels of H2S exhibited bright photoacoustic and T1-weighted MRI in tumor sites compared to healthy tissues in the same mouse  for modulation and imaging of hypoxia in head and neck squamous cell carcinomas . In vivo MRI ability of the nanocomposite was evaluated using a 4T1 tumor-bearing mouse. The results showed that the tumor location exhibited enhanced images relative to the pre-injection image  are released, enabling Fe3O4 to interact with the water protons (T2 relaxation enhancement). The ability to use the reported material was demonstrated via effective passive tumor targeting for T1- and T2-weighted MRI in tumor-bearing mice , the 19F-MRI signal was measured and quantified using an external standard (25 mM of NaPF6). A proof of concept of this approach was demonstrated in vitro by acquiring 1H- and 19F-MRI of four samples containing the polymeric material at different concentrations and pH values Dual-mode radiometric probes to overcome limitations of individual imaging modalities, for example, concentration dependency of contrast agents in MRI. Dual-mode imaging enables the integration of complementary advantages while minimizing the limitations of each method; however, these advantages and limitations are unique to instrumentation or the physics of methodology . The use of ratiometric probes is important to uniquely address some of the critical challenges associated with each imaging modality  and ratiometric probes enable obtaining precise information about environments and minimizes the chances of observing false negative and false positive results.(2)Dual-mode probes to detect relevant differences in physiological environments. Almost all reported probes are unable to detect the early stages of hypoxia. For example, pH-sensitive probes undergo stimuli-responsive changes in the probe when the pH is usually about 6.0–6.5. If a probe could detect a pH value slightly less than 7.4, such tissues could be flagged as potential cancer early, although with a secondary confirmation to avoid false positives. In PET imaging, most tracers detect tumors when the oxygen concentration is less than the clinically relevant hypoxic level (<10 mmHg). Detection of early stages of hypoxia can help maximize the survival rate of cancer patients; therefore, the development of dual-mode probes that are responsive to small changes in physiological conditions is highly desirable in future biomedical applications.(3)Increased biocompatibility of dual-mode probes for clinical imaging. Functionalizing dual-mode probes to increase biocompatibility, target solubility (water or lipid), and target specificity while minimizing toxicity is needed to use dual-mode probes in clinical imaging. Furthermore, imaging agents should be developed to remain active long enough that the instrument can detect the probe within an imaging-relevant time frame but short enough to clear within a reasonable amount of time.(4)Development of multiple stimuli-responsive probes. Reported dual-mode probes to date are responsive to specific changes associated with tumor microenvironments. However, the development of probes that are capable of simultaneously detecting more than one stimulus is likely to provide a more precise and accurate characterization of hypoxic tumors.(5)Integration of dual-mode diagnostic agents with therapeutic agents to detect and treat cancer simultaneously. New physical and chemical theories have been used in synthesizing activity-based sensing probes for tumor imaging and therapy ,400. Thi(6)The use of multiple imaging modalities to obtain accurate information about tumor microenvironments. Several multi-modal imaging probes have been reported to measure hypoxia. The inclusion of extra modalities, when selected for a specific purpose, can further overcome the limitations of using only two modalities; however, care must be shown to reasonably determine the utility of these extra modalities.In this review, advances in the sensitive detection of cancer using dual-mode imaging systems and a diverse group of responsive probes for hypoxia, oxidative stress, pH, and dysregulation of redox homeostasis are described. However, despite recent progress, most attempts are still in the preclinical stage and are not used in clinical imaging due to several challenges. Therefore, innovative strategies are needed to fill the gap between benchtop research and clinical imaging. Some aspects that are likely to be useful in future research to bridge laboratory research and tumor hypoxia imaging in cancer patients include the following:In summary, hypoxia is a critical medical condition that needs to be diagnosed in its early stages to increase the survival chances of patients and to develop new treatments for diseases. Dual-mode imaging of hypoxia has the potential to enable accurate diagnosis while overcoming the limitations of single-mode imaging techniques. Existing methods are still not capable of capturing subtle changes in tissue environment that help to identify tumors in their early stages. As a result, there is an opportunity to incorporate new ideas into existing methods and use them in clinical imaging. As opposed to its advantages, dual-modality imaging of hypoxia has limitations as well. For example, the necessity to use more than one imaging mode can be costly and time-consuming, and the design and synthesis of multimodal probes can be challenging. We expect that this review article will provide guidance for future development of imaging science focused on understanding and imaging hypoxia using more than one imaging modality at a time.


Tumour immune microenvironment heterogeneity is prevalent in numerous cancers and can negatively impact immunotherapy response. Immune heterogeneity and evolution in gastroesophageal adenocarcinoma (GEA) have not been studied in the past.Together with a multi-region sampling of normal, primary and metastatic tissues, we performed whole exome sequencing, TCR sequencing as well as immune cell infiltration estimation through deconvolution of gene expression signals.We discovered high TCR repertoire and immune cell infiltration heterogeneity among metastatic sites, while they were homogeneous among primary and normal samples. Metastatic sites shared high levels of abundant TCR clonotypes with blood, indicating immune surveillance via blood. Metastatic sites also had low levels of tumour-eliminating immune cells and were undergoing heavy immunomodulation compared to normal and primary tumour tissues. There was co-evolution of neo-antigen and TCR repertoire, but only in patients with late diverging mutational evolution. Co-evolution of TCR repertoire and immune cell infiltration was seen in all except one patient.Our findings revealed immune heterogeneity and co-evolution in GEA, which may inform immunotherapy decision-making.The online version contains supplementary material available at 10.1007/s10120-022-01324-7. Gastroesophageal adenocarcinoma (GEA) is an aggressive cancer that can metastasize to various sites . PatientThe T cell receptor (TCR) repertoire and immune cell infiltration are two important aspects of the TIME. TCR repertoire represents the entire collection of TCR sequences and their abundances in an individual . SeveralInfiltration of various types of immune cells into the tumour microenvironment has been shown to play key role in tumour development. For example, neutrophils, macrophages and CD4 + regulatory T cells support tumour development, whereas CD8 + cytotoxic T cells, natural killer cells and gamma-delta T cells actively eliminate tumours . Tumour These previous GEA studies, however, did not assess heterogeneity in TIME. TIME heterogeneity is prevalent in human cancers, and impacts both prognosis and immunotherapy response –18. For Lack of immune heterogeneity studies in GEA stems from difficulty in acquiring specimens from multiple regions of normal tissue, primary tumour, and metastatic tissue from various sites from the same patient. However, we have access to a multi-region sampling of normal, primary, and metastatic tumour tissue, and conducted the first systematic analysis of immune heterogeneity and evolution in patients with metastatic GEA who underwent emergency surgery due to active tumour-related gastric bleeding and gastric antrum obstruction. We performed whole exome sequencing, TCR sequencing as well as immune cell infiltration estimation through deconvolution of gene expression signals, to examine the intra-tumour, intra-patient, and inter-patient tumour immune microenvironment heterogeneity in patients with GEA. Findings here may aid in the prediction of immunotherapy benefit and inform decision-making in the future.N = 2) and gastric antrum obstruction (N = 3) were enrolled in this study. The patients’ primary gastric tumour tissues, adjacent normal gastric tissues and metastatic tumour tissue from lymph node, the peritoneum, liver and uterine attachment were retrieved at time of surgery. Tissue samples were immediately snap-frozen after resection or biopsy and stored in liquid nitrogen awaiting nucleic acid extraction. Two to three samples from normal and primary gastric tumour tissue were taken to assess spatial heterogeneity. Blood samples were collected before surgery from each patient. White blood was immediately transferred to cryogenic tubes for storage at − 80℃ awaiting germline DNA extraction.Five patients with metastatic GEA who were initially admitted to the Department of Gastric Surgery , from November 2019 to July 2020, for emergency surgery due to active tumour-related gastric bleeding (DNA from tissue samples and blood samples were extracted using a DNeasy Blood & Tissue Kit (Qiagen). Purified DNAs were quality checked using Nanodrop 2000 for A260/280 and A260/A230 ratios (Thermo Fisher Scientific). All DNA samples were quantified by Qubit 3.0 using the dsDNA HS Assay Kit (Life Technologies). Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems). Target enrichment was performed using the xGen Exome Research Panel and Hybridization and Wash Reagents Kit  according to the manufacturer’s protocol. Libraries were subjected to PCR amplification with KAPA HiFi HotStart ReadyMix (KAPA Biosystems). The purified library was quantified using the KAPA Library Quantification Kit (KAPA Biosystems), and its fragment size distribution was analyzed using a Bioanalyzer 2100 (Agilent). Sequencing was performed on Illumina HiSeq4000 platform using PE150 sequencing chemistry  to a mean coverage depth of 150X for tissue samples, and 60X for matched normal control blood samples.FASTQ file quality control was performed using Trimmomatic , where NTreeomics 1.7.3 , a well-DNA isolated from tissue and blood was subjected to T cell receptor (TCR) sequencing. In brief, the CDR3 region of TCR-beta chain was amplified with custom multiplex PCR primers (51 V-forward and 14 J-reverse primers) to capture all possible V(D)J combinations. Library was constructed and sequenced using the Illumina MiSeq platform to 10 M reads per sample. FASTQ file quality control was performed using Trimmomatic , where NDiversity measures assess the distribution of clonotype abundances within a sample’s TCR repertoire. We assessed 4 different diversity measures: richness, clonality, Shannon entropy and proportion of highly expanded clones. Richness represent number of unique clonotypes in a sample. Richness was normalized for read depth and relative T cell fraction  to allow fair comparison between samples. Clonality was defined as 1-Peilou’s evenness. High clonality (low evenness) implies skewing of clonotype abundance, whereas low clonality (high evenness) implies all clonotypes are nearly equally distributed. Shannon entropy is a measure that accounts for both richness and evenness . Highly Total RNA from tissue samples were extracted using an RNeasy Mini Kit (Qiagen). RNA purity was checked using Nanodrop 2000 for A260/280 and A260/A230 ratios (Thermo Fisher Scientific). All RNA samples were quantified by Qubit 3.0 using the RNA BR Assay Kit (Life Technologies). RNA integrity was assessed using Bioanalyzer 2100 (Agilent). RIN value (RNA integrity number) higher than 6.5 was required.RNA sequencing libraries were prepared using KAPA Stranded RNA-Seq Kit with RiboErase (KAPA Biosystems). Briefly, rRNA was first depleted with RiboErase, followed by DNase digestion for DNA removal. Purified RNA was subjected to first strand cDNA synthesis, followed by second strand synthesis with dUTP marking for strand-specificity. This is followed by A-tailing, adapter ligation and library amplification. Final library was quantified using KAPA Library Quantification Kit (KAPA Biosystems), and its fragment size distribution was analyzed using a Bioanalyzer 2100 (Agilent). Sequencing was performed on the Illumina HiSeq4000 platform using PE150 sequencing chemistry  to an average of 60 M reads per sample.FASTQ file quality control was performed using Trimmomatic , where Nhttp://cibersort.stanford.edu/) and ran locally. In brief, each sample has its gene expression profile. This profile is a linear combination of the known gene expression signature matrix of 22 types of immune cells and the relative fraction of those immune cells. CIBERSORT uses deconvolution through support vector regression to estimate the relative fraction of 22 types of immune cells.Relative infiltration level of 22 types of immune cell was estimated for each sample with CIBERSORT , using gLevel of immunomodulation was assessed as per a previous study by Thorssen et al. Gene expression levels in transcript per million (TPM) of 70 immunomodulator genes were assessed . These gCytolytic activity was calculated as the geometric mean of gene expression levels of GZMA and PRF1 as previously described .Neoantigen prediction is based on an assessment of the binding potential of mutation-derived peptide against patient’s MHC-I and MHC-II. HLA typing was performed using ATHLATES  and WES P value < 0.05 was seen as having statistically significant co-evolution.Evolutionary relationships between primary and metastatic samples for each patient were constructed using hierarchical clustering based on Jaccard distance for neoantigen and TCR repertoire and Euclidean distance for immune cell infiltration. Co-evolution analysis involved the comparison of hierarchically clustered dendrogram trees between neoantigen, TCR repertoire and immune cell infiltration using cophenetic correlation from the dendextend package  as previSeveral types of heterogeneity analysis were conducted. Pairwise comparisons were all made within each patient. Comparisons for all patients were represented with a boxplot. Intra-primary represents heterogeneity between different samplings from the same primary tumour. Inter-normal represents samples from spatially separated normal tissue within a patient. Inter-metastasis represents samples of spatially separated metastatic tissue. Three metastatic sites would mean 6 comparisons. Inter-primary-normal, inter-primary-metastasis, inter-metastasis-normal represent heterogeneity from the respective pairs of tissue categories. Euclidean distance was used to score the heterogeneity between samples. If two samples share a low amount of infiltrating immune cell abundance, the Euclidean distance would be greater, meaning that the two samples are more heterogeneous to each other.Gene expression levels of well-established immunotherapy biomarkers including cytolytic activity (CYT), PD-L1 (PDCD1LG1), PD-L2 (PDCD1LG2), PD-1 (PDCD1), CD39, CD8A, CD8B and CD4 were z-score normalized across different samples within each patient. Tumour mutational burden (TMB) was counted by summing all non-synonymous alterations. CIS (chromosome instability score) was defined as the proportion of the genome with aberrant (purity-adjusted segment-level copy number ≥ 3 or ≤ 1) segmented copy number. OncoKB  v1.24 pap < 0.05 was considered to indicate a significant difference. False Discovery Rate was used for multiple testing correction. All statistical analyses were performed with R packages (v.3.5.3).Non-parametric Wilcoxon’s rank-sum test was used to assess the differences between groups. N = 2) or gastric antrum obstruction (N = 3), had negative Epstein–Barr virus (EBV) status based on Epstein–Barr virus-encoded small RNAs (EBER) in situ hybridization, were microsatellite stable and did not have human leukocyte antigen loss of heterozygosity (HLA-LOH). Only one patient was HER2 positive  years old. All patients had stage IV GEA, underwent palliative surgery due to tumour-related bleeding  were made for heterogeneity analysis. A schematic of how the comparisons were made is shown in Fig. In terms of TCR heterogeneity, it was highest amongst metastatic samples (inter-metastasis), with an average only 4.67% of TCR clonotypes were shared, as well as between primary tumour and metastatic samples (inter-primary-metastasis) with 4.26% shared, and between normal gastric tissue and metastatic samples  with 3.99% shared  Fig. B on aveC and higWe further divided the metastasis group into the lymph node and other metastasis. The purpose of this was to determine how lymph nodes, which filter T cells regularly, may present antigen in a different manner before and after developing metastasis. We found that the concordance of the TCR repertoire of lymph node metastasis and the primary tumour is lower than other metastases and primary tumour . Based on this, we deduce that this is due to the circulating T cells being captured during sample collection. In this case, a portion of unrelated TCRs would be captured, thus resulting in the observed diverse repertoire.To gauge the amount of circulatory immune surveillance in metastatic GEA, we assessed TCR clonotype sharing between blood and normal tissue, primary tumour and metastasis. There is very little TCR clonotype sharing between blood and normal, primary or metastatic tissue Fig. F. Only ~We also assessed T cell receptor’s VJ segment differential usage between blood, normal gastric tissue, primary tumour and metastatic tissue. Highest number of differential VJ usage was found between blood and the other tissue types . This is expected since we saw very little TCR clonotype sharing between blood and other tissue types. Normal gastric tissue was also enriched in many VJ segments not seen in metastatic tissue . Most interestingly, one VJ segment (TRBV25-1 TRBJ2-5) was enriched in the primary tumour compared to metastatic tissue .Together these results suggest a highly heterogeneous T cell population among metastatic sites. T cells are being supplied to metastatic sites via blood. However, these T cell clones are not yet expanded and not active in metastasis.Next, we assessed immune cell infiltration levels and heterogeneity within and between normal, primary tumour and metastatic tissues using CIBERSORT , which qWe also assessed immunomodulation via the expression of 70 immunomodulatory genes as previous described . On averTogether these results show a highly heterogeneous immune cell infiltration landscape among metastases. Lower infiltration of tumour eliminating immune cells in metastatic tissue, and a heavily immunomodulated metastatic environment.P value (0.08)] ]  Fig. D. This iLastly, we assessed expression levels of well-known immunotherapy biomarkers across samples. We found these immunotherapy biomarkers are heterogeneous across samples Fig. . This spHeterogeneity of tumour immune microenvironment complicates the assessment of immunotherapy biomarkers and can lead to non-uniform response to immune checkpoint inhibitors (ICIs). In this study, we performed multi-region sampling of normal gastric tissue, primary tumour and metastatic tissue from 5 treatment naïve patients. Using a multi-omics approach, we analyzed T cell receptor repertoire and immune cell infiltration, along with their heterogeneities both within and between the tissue samples. We also compared evolutionary trajectories between the tumour genome and immune microenvironment.We found that, when compared to normal and primary gastric tumours, metastasis in GEA had a pro-tumour immune microenvironment. Metastasis had high richness but low TCR clonality, which represents a lack of dominant and potentially tumour-targeting T cell clones . Previous studies have found those with high TCR clonality prior to anti-PD-1 therapy had longer progression-free survival in metastatic melanoma , and betFor both T cell receptor repertoire and immune cell infiltration, there was high heterogeneity amongst metastatic samples compared to the relatively homogeneous immune landscape of primary tumour and normal gastric samples . Same metastatic organs also exhibited drastically different levels of immune cell infiltration, as well as TCR diversity measures. Diverse levels of immune cell infiltration across metastases were also seen in breast cancer . The divTo gauge the interaction between tumour genome changes and response of the host immunity, we compared the evolution of neo-antigen, TCR repertoire and immune cell infiltration. Neo-antigens are mutant peptides capable of triggering T cell clonal expansion . We founFinally, we found the expression of immunotherapy biomarkers, as well as TMB and CIS, were heterogeneous. Similar region-to-region heterogeneity has also been observed for PD-1 and PD-L1 expression in NSCLC . This heUsing a multi-region, multi-omics approach, this study revealed a heterogeneous immune landscape in metastatic GEA with mutational-immune co-evolution in certain patients. These findings may have implications for immunotherapy decision-making.Supplementary file1 (PDF 5872 KB)Below is the link to the electronic supplementary material.


After the first and second vaccination, ELISpot responses towards the glycoprotein E were significantly higher in males versus females . Multivariate analysis showed that shingles and sex both impacts significantly on VZV immunity. Whereas vaccination-induced changes could hardly be detected after stimulation with a whole VZV antigen, there was a significant increase in responses towards glycoprotein E after vaccination (p < 0.005). These data indicate that vaccination with Shingrix™ augmented cellular, VZV-specific immunity in HSCT recipients. Shingles and male sex could both be identified as factors leading to increased immunity.Hematopoietic stem cell transplant (HSCT) recipients have a high risk of developing primary varicella-zoster virus (VZV) infection and reactivation. VZV vaccination may prevent infection and reactivation. In the current study, recipients of allogeneic HSCT  were vaccinated with adjuvanted, recombinant zoster vaccine Shingrix™, which contains the VZV glycoprotein E. Cellular immunity against various VZV antigens was analyzed by interferon-gamma ELISpot. Peripheral blood mononuclear cells (PBMC) of recipients with versus without prior shingles ( Varicella-zoster virus (VZV), a double-stranded DNA virus, is a member of the herpesvirus family, which causes varicella/chickenpox after primary infection and zoster/shingles after reactivation. Following centripetal axonal transport, circular viral DNA persists in neurons of dorsal root and cranial nerve ganglia, where it can remain quiescent for decades . As all If varicella or shingles occur after allogeneic HSCT, passive immunization with anti-VZV hyperimmunoglobin (Ig) within 96 h of exposure is recommended .TM, GlaxoSmithKline Biologicals S.A., Rixensart, Belgien) is recommended, especially from the age of 60 and for individuals with immunodeficiency [TM could reduce the risk of contracting shingles during lifetime in the general population from 33% to 3% [TM and 130 who received placebo, this study reported a lower rate of suspected cases of shingles in vaccinees (3 vs. 7 suspected cases). Currently, there is no data if vaccination with ShingrixTM could induce specific cellular immunity in allogeneic HSCT recipients, which was addressed in the present study.In Germany, the United States and many other countries, a live attenuated vaccine is licensed and its use is recommended for vaccination against primary infection ,12. Moreficiency . It cont3% to 3% . Moreove3% to 3% . ConsideTM, in which T cell immunity against VZV-specific antigens was analyzed. We stimulated the cells with inactivated whole virus, native glycoprotein and peptides of glycoprotein E (gE), the most abundant and immune-dominant glycoprotein of VZV [We report on a HSCT cohort vaccinated twice with Shingrixn of VZV . Becausen of VZV . MoreoveTM , up to three times after the first vaccination (51 samples) and up to 6 times after the second vaccination (100 samples). We considered all these samples to correlate ELISpot responses to the different VZV antigens. Sequential analyses were performed in 54 patients. It should be noted that the time courses presented in the manuscript only consider the time point closest to vaccination, i.e., one value before vaccination and one after the first and second vaccination, respectively.For comparison, we performed sequential analyses in four age-matched, healthy controls .The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the University Hospital Essen, Germany (19-9040-BO). Informed consent was obtained from all subjects involved in the study.TM contains 50 µg of the adjuvanted, recombinant VZV gE antigen, produced in immortalized ovarian cells of the Chinese hamster (CHO cells) [Quillaja saponaria Molina, fraction 21 (QS-21) and 50 µg 3-O-desacyl-4′-monophosphoryl lipid A (MPL) from Salmonella minnesota. ShingrixTM is licensed for the prevention of shingles and postherpetic neuralgia in adults ≥50 years of age [The subunit vaccine ShingrixO cells) . It is as of age . Vaccina® ELISpot kit, Mikrogen GmbH, Neuried, Germany; formerly Lophius Biosciences GmbH, Regensburg, Germany). Cultures of 200,000 freshly isolated PBMC were incubated without and with VZV antigens in 150 µL of AIMV medium  at 37 °C for 19 h. Stimulation with the T cell mitogen phytohemagglutinin  served as a positive control. Colorimetric detection of cytokine secreting cells was performed according to the manufacturer’s instructions. Spot numbers were analyzed by an ELISpot plate reader . VZV-specific spots were determined as stimulated minus non-stimulated (background) values (spots increment). Of note, the negative controls reached a median value of 0, a mean of 0.38 spots and a standard deviation of 0.94 spots. The positive control with PHA indicated that all results included in this study were valid (median 417 spots (range 59–>600).A total of 9 milliliters of heparinized blood was collected, and peripheral blood mononuclear cells (PBMC) were separated within 24 h after collection by Ficoll gradient centrifugation. Numbers of PBMC were determined by an automated hematology analyzer . To assess VZV-specific cellular immunity, we performed IFN-γ ELISpot assays, using freshly isolated PBMC and two protein antigens and a peptide pool as stimuli. In parallel experiments, we applied a whole VZV antigen , a native VZV glycoprotein  and a gE peptide pool . The whole VZV antigen is produced from human tissue cell cultures infected with VZV and is lyophilized after inactivation and addition of a stabilator. One vial of lyophilized whole VZV antigen was diluted in 2 mL or 8 mL of aqua distillata (dilution 1:2 or 1:8) and 10 µL of the diluted antigen was used per cell culture. The gE peptide pool contains 153 peptides derived from a peptide scan (15-mers with 11 aa overlap) through envelope protein (Swiss-Prot ID: P09259) of VZV strain Dumas. The production of IFN-γ was determined using pre-coated ELISpot plates and a standardized detection system . The impact of shingles and sex on ELISpot responses was analyzed by multivariate analysis . If not otherwise stated, median values are indicated. The results were considered significant at p < 0.05.Data were analyzed using GraphPad Prism 8.4.2.679  or IBM SPSS Statistics version 25 . ELISpot responses towards different VZV antigens, used at various concentrations, were compared by One-way ANOVA, using a mixed effects model and the Holm–Sidak’s multiple comparisons test. Time courses of ELISpot responses in patients with and without shingles were compared by Two-way ANOVA using the Bonferroni post-test. Results prior to and post vaccination were compared by Wilcoxon matched pairs test, data in two or more independent groups by the Mann–Whitney n = 236 samples)  a,b. The samples) a. For thsamples) b.p values of 0.02 or 0.004, respectively. Moreover, we calculated how many patients showed an at least twofold increase in ELISpot responses, which was defined as criterion for the effectiveness of the vaccination. Considering whole VZV antigen at a dilution of 1:2 or 1:8, 6 out of 30 patients (20%) or 12 out of 28 (43%) fulfilled this criterion. Considering gE peptides at a concentration of 1 and 0.5 µg/mL per peptide, the respective numbers were 6 out of 10 patients (60%) or 10 out of 11 (91%). Thus, changes may be detected with higher sensitivity when lower antigen concentrations were used.We compared VZV (gE)-specific immunity in HSCT recipients tested prior to and post 2nd vaccination with the adjuvanted, recombinant zoster vaccine Shingrix™ . Of noteTwo patients could be tested very early after the onset of shingles . In the p < 0.005). VZV gE-specific ELISpot responses prior to and post vaccination were compared between HSCT patients and four healthy controls. Prior to vaccination, VZV-specific ELISpot responses towards the gE peptide pool were comparable. However, after the first and second vaccination with Shingrix™, cellular responses were 4.8-fold and 1.8-fold higher in the age-matched healthy controls vs. HSCT patients .Furthermore, considering the first sample of each HSCT patient tested prior to vaccination and after the first and second vaccination, we analyzed if gE-specific ELISpot responses increased after vaccination . Of noteIn HSCT patients with vs. without prior shingles, ELISpot responses towards a whole VZV antigen were approximately twofold higher prior to vaccination  a,b. RespIn contrast, in HSCT patients with vs. without prior shingles, ELISpot responses towards the gE peptides were similar prior to vaccination and responses increased after the first and second vaccination. In patients with prior shingles, the number of spots increment increased 5.3- and 6.7-fold, in patients without prior shingles 1.5- and 3.7-fold (1 µg/mL per peptide) c. Similap ≤ 0.02) (p = 0.02) (p = 0.01) (p = 0.07) and 4 vs. 17 (p = 0.02)  c,d. Afte = 0.02) c and 1 v = 0.01) d, respec = 0.02) c,d.However, ELISpot responses did not correlate significantly with age, underlying disease, acute or chronic GvHD, myeloablative preparative regimen, treatment with anti-thymocyte globulin or stem cell source . Moreover, we could not observe a significant correlation with the interval between transplantation and vaccination, the interval between first and second vaccination, and the interval between first or second vaccination and blood sampling, respectively.p = 0.03) and sex (p = 0.04).The impact of shingles and sex on ELISpot responses was then examined by multivariate analysis . Findingr = 0.36–0.54).Spearman analysis indicated that ELISpot responses towards the peptide pool of gE and the other VZV antigens were positively correlated in the majority of cases. As a first step, we analyzed three VZV antigens, used at different concentrations in seven HSCT patients . Correlan = 236 samples). Correlation coefficients were similar, and all correlations were highly significant (p < 0.0001)  than responses towards various VZV antigens . Moreover, correlations between responses to gE and whole VZV antigen were stronger after first and second vaccination than prior to vaccination.We then analyzed the total cohort ( 0.0001) . As exper = 0.68–0.91). (p < 0.005).In addition, we performed Spearman analyses for ELISpot results obtained at different time points. The respective correlation analyses showed moderate to strong correlation (8–0.91). . ELISpotTM augmented cellular immunity in allogeneic HSCT recipients, which had not yet been shown. A comparative analysis of various VZV antigens showed that gE may be more suitable than whole VZV antigens for measuring vaccine-induced VZV gE-specific immunity. We observed that cellular responses towards gE increased significantly after vaccination, whereas responses to whole VZV did not. gE is the most abundant and immune-dominant glycoprotein expressed on the surface of VZV-infected cells [The current study indicates that vaccination with Shingrixed cells  and consed cells . As comped cells . Moreoveed cells  and quaned cells . Similared cells . This grIn a cohort of 32 kidney transplant recipients, there are already data on T cell immunity after vaccination with Shingrix™ . ImmunitIn autologous HSCT recipients ≥18 years of age, a vaccination efficacy of 68% has been reported, summarizing results of three studies . HoweverOur study has several limitations. The major point is that there was variation in the timing of vaccinations and blood sampling. The patients were not called in especially for the study and most of them presented at our site only rarely due to the COVID-19 pandemic. However, there was no special evaluation scheme depending on subgroups. Moreover, a shortage of the vaccine may have extended the interval between the two vaccinations. To partly address this point, we presented a subset of data separately . We showIn the current study on adult allogeneic HSCT recipients, we show that cellular immunity against gE of VZV increased after vaccination with Shingrix™. Furthermore, we identify prior shingles and male sex as factors leading to increased VZV-specific immunity. Whereas the influence of prior shingles was expected at first glance, the influence of sex is less clear. VZV reactivation leads to an in vivo expansion of VZV-specific T cells, which may be further “boostered” after vaccination. Previous literature indicates that immune responses in females and males may differ, for example the concentration of cytokines or vaccine antibodies ,34,35,36In allogeneic HSCT recipients, vaccination with the adjuvanted, recombinant vaccine Shingrix™, which contains the VZV gE, led to a significant increase in cellular in vitro responses towards VZV gE. As compared to the controls, cellular responses were weaker. The current data indicate that patients with prior shingles and males exhibit higher VZV-specific IFN-γ responses. It needs to be clarified whether the higher ELISpot responses correlate with better protection against VZV infection and reactivation.