This regulation controls the formation of melanotic masses and developmental growth. Overall, our findings provide valuable insights into melanotic mass formation and developmental growth as well as the regulatory network of the JNK and JAK/STAT signaling pathways.In animals, including humans and flies, blood cells play crucial roles in various biological processes, such as immune response and normal development. The abnormal regulation of hematopoiesis in flies results in the formation of melanotic masses, which are also known as melanotic tumors. However, the detailed mechanisms underlying this process are not fully understood. In this study, we found that the upregulation of miR-274-5p, a small non-coding RNA, activates the JNK and JAK/STAT signaling pathways by suppressing the expression of miR-274 in the fat body resulted in the formation of melanotic masses. Moreover, abnormal activation of the JNK and JAK/STAT signaling pathways was linked to these consequences. In addition to this defect, miR-274 overexpression in the larval fat body decreased the total tissue size, leading to a reduction in body weight. miR-274-5p was found to directly suppress the expression of found-in-neurons (fne), which encodes an RNA-binding protein. Similar to the effects of miR-274 overexpression, fne depletion led to melanotic mass formation and growth reduction. Collectively, miR-274 plays a regulatory role in the fne–JNK signaling axis in melanotic mass formation and growth control.The hematopoietic system plays a crucial role in immune defense response and normal development, and it is regulated by various factors from other tissues. The dysregulation of hematopoiesis is associated with melanotic mass formation; however, the molecular mechanisms underlying this process are poorly understood. Here, we observed that the overexpression of  Drosophila, hematopoiesis occurs in two different waves: in the head mesoderm of early embryos and the lymph glands of larvae [Drosophila prohemocytes terminally differentiate into three types of blood cells: plasmatocytes (phagocytosis), crystal cells (melanization), and lamellocytes (encapsulation) [Blood cells play important roles in the immune response against invading pathogens and in the normal development of metazoan ,2. In Drf larvae ,4,5. Durulation) ,7. Lamelulation) . Melanizulation) . This heDrosophila, melanotic mass formation, a conspicuous cellular response, can be induced via abnormal immune responses, such as lymph gland overgrowth and the massive differentiation of lamellocytes [Drosophila immune deficiency (IMD)/Relish, and Ras/mitogen-activated protein kinase (Ras/MAPK) pathways [Drosophila, binding of the cytokine, Unpaired (Upd), to the Domeless (Dome) receptor induces receptor dimerization and activation of the JAK Hopscotch (Hop in Drosophila). Activated Hops phosphorylate STAT proteins, allowing them to form dimers and translocate into the nucleus to regulate the expression of target genes, such as the Suppressor of cytokine signaling at 36E (Scocs36E) [Upds, which is a ligand of the JAK/STAT pathway [In llocytes ,9. The fpathways . The JAKcocs36E) . Loss ofcocs36E) . The act pathway . These c pathway ,11. This pathway ,11. FurtDrosophila undergoes growth until a distinct stage, known as the larval-to-pupal transition, which is triggered by the ecdysone steroid hormone. The final body size of Drosophila is influenced by the growth rate during this stage and the duration of growth [slimfast (slif), which encodes an amino acid transporter, leads to a growth defect through the target of rapamycin (TOR) signaling pathway [f growth . Under tf growth . These g pathway . HoweverDrosophila melanogaster [Drosophila miR-279 is involved in ovarian cell fate and circadian behavior by regulating stat92E and upd, respectively [found-in-neurons (fne) gene encodes an RNA-binding protein as one of the three paralogs  of the ELAV gene family, and it is primarily expressed in neuronal tissues in Drosophila [fne is associated with several biological processes, such as mushroom body development, male courtship performance, and synaptic plasticity [fne in non-neuronal cells remain unknown.MicroRNAs (miRNAs) are small non-coding RNAs (~21 nucleotides in length) that post-transcriptionally suppress gene expression by inducing RNA degradation and/or translational repression . After tnogaster . Individnogaster . Throughnogaster . In partectively . Additioectively . Howeverosophila . Accordiasticity ,25. In aasticity . The cytasticity . HoweverDrosophila, glia-derived miR-274 is known to play a role in regulating the growth of synaptic boutons and tracheal branches through the targeting of Sprouty [Drosophila under pathogen bacteria, Micrococcus luteus infection conditions [Drosophila miR-274 in larval fat bodies in terms of melanotic mass formation and developmental growth. We found that miR-274 overexpression results in the activation of the JNK and JAK/STAT signaling pathways, which are closely associated with the observed phenotypic consequences. Furthermore, we revealed that this regulation of miR-274 was mediated by the RNA-binding protein Fne, which is a biologically relevant target of miR-274. Overall, our findings suggest that miR-274 plays a crucial role in regulating the fne–JNK signaling axis under its upregulated conditions, which in turn affects melanotic mass formation and developmental growth.In nditions . HoweverGAL4/UAS system. The following fly lines from the Bloomington Drosophila Stock Center were used: 1118w (BL5905), Cg-GAL4 (BL7011), ppl-GAL4 (BL58768), UAS-LUC-miR-274 (BL41172), and TRiPUAS-fne-RNAi (BL28784).All flies were grown at 25 °C on standard cornmeal/agar medium under noncrowded conditions. Transgenic overexpression studies were performed using the All larvae and flies were maintained on standard cornmeal/agar media. Melanotic masses were analyzed in wandering third-instar larvae and adult flies as previously described . For quaAfter the eggs were laid on standard cornmeal/agar media at 25 °C, three vials containing eggs from each genotype were transferred to a 29 °C incubator. More than 130 pupae were analyzed for each genotype at 15 d AEL, and the rate of empty puparia in each vial was calculated to determine the eclosion rate.g. Fat bodies were dissected in cold phosphate-buffered saline (PBS) from wandering third-instar larvae in cold PBS. To collect hemolymph-containing hemocytes, we used a previously described protocol with some modifications [Total RNA was purified from S2 cells, larval fat bodies, and adult heads using the TRI Reagent , according to the manufacturer’s instructions. TRI Reagent BD (Molecular Research Center) was used for RNA isolation from larval hemocytes. The following protocol was used for each sample: S2 cells were harvested via centrifugation for 5 min at 350× ications . BrieflyE. coli poly(A) polymerase , and the polyadenylated RNAs were reverse-transcribed using M-MLV Reverse Transcriptase (Enzynomics) and an miR-RT-adapter-primer  or BioFACTTM 2× Taq PCR Master Mix (BIOFACT). PCR products were loaded into 2.5% agarose gel and then stained using SYBRTM Safe . The band images were captured on a blue light transilluminator . The primer sequences used for semi- or quantitative-RT-PCR are listed in miRNA quantification was performed using a PCR-based method, as previously described . In brier-primer . To measWestern blot analysis was performed as previously described . The folGroups of 8–10 adult flies of each genotype (3–5 days old) were transferred to new 1.5 mL tubes and weighed using an analytical balance . Four biological replicates per genotype and sex were analyzed.The left wings of adult female (5 days old) were used for wing analysis. After capturing images using a stereomicroscope (Olympus), the relative wing size was measured using ImageJ software version 1.53 . The winTo capture an image of the whole larval fat body, the fat body was dissected from five wandering third-instar larvae of each genotype and photographed using a stereomicroscope (Olympus).Phalloidin staining was performed as previously described . BrieflyDrosophila S2 cells were maintained at 25 °C in Schneider’s insect medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum  and 100 U/mL penicillin–streptomycin (Welgene).fne 3′-UTR was amplified by PCR. The amplified DNA fragment was then inserted downstream of the Renilla luciferase in the psiCHECK-2 vector . For the mutant form of the fne 3′-UTR, site-directed mutagenesis was performed using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific), as previously described [miR-274-overexpressing construct, a DNA fragment containing pre-miR-274 was amplified by PCR and cloned into the pMT/V5-His A vector . Both the miRNA-expressing and luciferase reporter constructs were co-transfected using TransIT®-Insect Transfection Reagent . The activities of Renilla and firefly luciferase were determined using the Dual-Luciferase Reporter Assay System  48 h after miR-274 expression. The Renilla luciferase activity was normalized to the firefly luciferase activity. All primer sets for the reporter assay are listed in To generate plasmid constructs for the reporter assay, the genomic region of the escribed . To genemiR-274 exhibits dynamic changes during Drosophila development [Drosophila, we employed Cg-GAL4 to overexpress miR-274 . Cg-GAL4 is known to be active in fat bodies [Cg>miR-274 larvae, the expression of miR-274-5p, the major strand of miR-274, was significantly upregulated , which is a marker gene of lamellocytes [Itgbn was significantly higher in the hemocytes of Cg>miR-274 larvae than in the control  mRNA expression [Cg>miR-274 larvae, we sought to determine whether the expression of InR mRNA is changed in the fat body by miR-274 overexpression. As expected, the expression of InR mRNA was reduced in the fat body of Cg>miR-274 larvae compared with the controls  compared to Cg/+ control flies  that encodes an RNA-binding protein associated with the regulation of mRNA processing, such as splicing and alternative polyadenylation (APA) of mRNA [fne 3′-UTR: one conserved site (miR-274-5p BS1) and one poorly conserved site (miR-274-5p BS2)  A, top.Drosophila, fne mRNAs are mainly expressed in the nervous system and exist as several isoforms with different lengths of the 3′-UTR [fne mRNA is also expressed in the larval fat body in addition to the nervous system. By semi-RT-qPCR using two different primer sets, we detected fne mRNA with a short and extended 3′-UTR in the fat body of wandering third-instar larvae and adult heads. Consistent with previous results [fne mRNA transcript with an extended 3′-UTR was detected in adult heads and was also expressed in the fat body  fused with wild-type fne 3′-UTR significantly decreased   E. Taken fne is suppressed by miR-274 as a direct target in Drosophila, we sought to determine whether the loss of fne leads to phenotypic consequences similar to the defects caused by miR-274 driven by Cg-GAL4. The previous RNAi screening study reported that RNA splicing-associated protein fne may be associated with melanotic mass formation [fne expression in the fat body using Cg-GAL4 (Cg>fne-RNAi)  A. Consisi larvae B, and 42ormation C. Melanoormation D. These fne causes an increase in the activity of the JAK/STAT signaling pathway in the larval fat body, similar to Cg>miR-274 larvae. The expression level of socs36E was significantly higher in the fat bodies of Cg>fne-RNAi larvae than in the control fat bodies , Rac1, and cryptocephal (crc). Thus, changes in the length of the 3′-UTR of these genes involved in the JNK or the JAK/STAT signaling pathway, which are regulated by Fne, may alter their expression level.In this study, we demonstrated that miR-274 activates the JNK–JAK/STAT signaling axis by targeting  reports , Fne may reports ,44. Baseof mRNAs , which mof mRNAs , target S2 cells . The idefne, which in turn induces the expression of upd3 in the fat body. Subsequently, Upd3 derived from the fat body may stimulate lamellocyte differentiation, triggering melanotic mass formation. Although we provided evidence of the association between miR-274 overexpression in the fat body and melanotic mass formation based on observations using Cg- and ppl-GAL4 drivers, we cannot completely exclude the possibility that miR-274 may regulate the JNK–JAK/STAT signaling pathway in blood cells in addition to the fat body. The Cg-GAL4 driver primarily activates the fat body but also drives gene expression in blood cells. [ppl>miR-274 larvae compared to Cg>miR-274 larvae. Thus, interactions between the regulatory networks of the fat body and hemocytes may synergistically influence melanotic mass formation.Our findings indicate that miR-274 activates JNK signaling by suppressing d cells. . FurthermiR-274 and fne were overexpressed and depleted, respectively. In addition to JAK/STAT signaling, JNK signaling is well known to play a pro-apoptotic role in cell death and negatively regulates insulin/IGF-like signaling in both mammals and Drosophila [Drosophila [fne, and developmental growth. In addition, a previous report described that knockdown of upd2 in the fat body, but not upd1 and upd3, leads to growth reduction by regulating the secretion of Drosophila insulin-like peptides in the insulin-producing cells [upd2 in the fat body may affect growth reduction.We noted a significant reduction in tissue size and activation of the JNK–JAK/STAT signaling pathway in the fat body when osophila ,48. The osophila ,50. Thesng cells . Therefopre-miR-274 is higher in the glia than in other tissues during the larval stage of Drosophila [fne, is primarily expressed in neuronal tissue in Drosophila [miR-274 and fne in the same tissues indicates that they are likely to maintain a regulatory network, which is similar to our findings in fat bodies. Based on our previous results, the expression of miR-274 is upregulated during the larval-to-pupal transition [miR-274 expression may alter the composition of the extended 3′-UTR of neuronal-specific genes by regulating the expression of fne during this developmental stage. Thus, future studies should investigate the perturbation of APA in neuron-specific genes during metamorphosis.According to previous reports, the expression of osophila . The expansition , which sfne mRNA and reporter activity with the fne 3′-UTR when miR-274 was overexpressed. miR-274-5p was found to negatively regulate the expression of fne at the transcript level by binding to at least one conserved site (miR-274-5p BS1) in the 3′-UTR of fne mRNA. However, the luciferase reporter activity suppressed by miR-274 was found to be partially restored when the region corresponding to the miR-274-5p seed was mutated in the miR-274-5p BS1. This result implies that other binding sites may exist for miR-274-5p in the fne 3′-UTR. The poorly conserved binding site (miR-274-5p BS2) in the fne 3′-UTR could be a potential regulatory binding site for miR-274-5p. Future studies could examine whether miR-274-5p targets miR-274-5p BS2 in the fne 3′-UTR.We observed a reduction in both fne-depleted larvae. Most Cg>miR-274 larvae exhibited a melanotic mass, whereas less than 50% of Cg>fne-RNAi larvae had black dots. The melanotic masses in the individual Cg>miR-274 larvae were numerous and larger than those in the Cg>fne-RNAi larvae. Such small differences between the lines linked in the regulatory network may arise from other genes affected by alterations in miR-274 or fne expression. TargetScanFly identified 173 potential miR-274-5p targets. Some of these targets, such as S-adenosylmethionine Synthetase (Sam-S) and slimfast (slif), might directly or indirectly affect the JNK and/or JAK/STAT signaling pathways. In addition, although miR-274-3p is not the predominant strand of miR-274, miR-274-3p might contribute to melanotic mass formation and/or growth. TargetScanFly identified numerous potential target genes of miR-274-3p. For example, GO term analysis using DAVID [Connector of kinase to AP-1, Menin 1, Protein phosphatase V, Ras-like protein A, Striatin interacting protein, alphabet, capping protein alpha, fiery mountain, flapwing, icarus, and pebbled) that can play negative regulators of the JNK signaling pathway.A relatively stronger formation of melanotic masses was observed in larvae overexpressing miR-274 than in ng DAVID ,53 reveaCollectively, our findings provide insights into the potential molecular mechanisms underlying these phenomena. Further studies should explore these mechanisms in detail to provide a more comprehensive understanding of the physiological processes.Drosophila miR-274 was found to play a regulatory role in the JNK and JAK/STAT signaling pathways by suppressing the expression of fne encoding an RNA-binding protein that functions in RNA processing, such as APA and alternative splicing. This regulatory network of miR-274–fne–JNK signaling in the larval fat body is associated with the formation of melanotic mass and developmental growth. Thus, our findings provide valuable insights into the molecular mechanisms underlying melanotic mass formation and growth. Furthermore, a better understanding of this regulatory mechanism will contribute to the development of novel ideas for regulating immune defense responses against invading pathogens and growth regulation.In this study, under its overexpression conditions,


Coronavirus disease 2019 in children presents with distinct phenotype in comparison to adults. Overall, the pediatric infection with a generally milder clinical course of the acute infection compared to adults still faces several unknown aspects. Specifically, the presence of a wide range of inflammatory manifestations, including multisystem inflammatory syndrome in children (MIS-C), myocarditis, and long COVID in the period after infection suggests a particular susceptibility of some children upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Albeit peculiar complications such as long covid are less frequent in children compared to adults, research on the relationship between inflammatory syndromes and SARS-CoV-2 is rapidly evolving.     Conclusions: new studies and findings continue to emerge, providing further insights into the underlying mechanisms and potential therapeutic strategies. In the present work, we revised current knowledge of the main factors accounting for such variability upon SARS-CoV-2 infection over the pediatric age group. Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019, our understanding of the virus and its associated clinical manifestations has rapidly evolved [Several uncertainties remain on reasons underlying the distinct and overall milder clinical course experienced by children in the acute phase of COVID19 when compared to adults or elderly , 3 . FewOther researchers also aimed at dissecting virological and immunological characteristics in SARS-CoV2 presenting with distinct symptomatology in children. We previously showed how the severity of COVID19 was not related to the extent and persistence of SARS-COV2 specific immunity; however, symptomatic children presented with higher pro-inflammatory cytokines (CXCL10 and CCL19) and longer time to SARS-COV2 clearance , 7. NoveMIS-C is uncommon but represents the most severe inflammatory syndrome observed in children and adolescents following SARS-CoV-2 infection. It typically manifests as persistent fever, along with gastrointestinal symptoms, rash, conjunctivitis, and cardiac abnormalities. Recent evidence suggests that MIS-C may result from an exaggerated immune response triggered by the virus, leading to systemic inflammation and endothelial dysfunction. Vanderbeck and colleagues highlighted the possible role of Notch signaling pathways which have emerged as important regulators of the immune system being at the crossroads of innate and adaptive immunity . BenamarIn addition to its respiratory and systemic effects, SARS-CoV-2 has been shown to induce a pro-thrombotic state, leading to an increased risk of venous and arterial thrombotic events even in childhood. The incidence of thrombotic events in hospitalized children was much higher in children with systemic inflammation in comparison with children with mild or asymptomatic SARS-CoV-2 infection , 15. TheCOVID-19-related neuroinflammation represents an area of growing concern and interest among healthcare professionals and researchers. Although pathogenic mechanisms of such complications have not been fully explored especially in the pediatric context, and SARS-CoV2 is not a classic neurotropic virus, recent findings show the impact of neurological disorders following COVID19 infection in adults , 19. Althttps://www.who.int/publications/i/item/WHO-2019-nCoV-Post-COVID-19-condition-CA-Clinical-case-definition-2023-1).A proportion of individuals recovering from acute COVID-19 continue to experience persistent symptoms, known as long COVID. These symptoms often include fatigue, dyspnea, cognitive impairment, and musculoskeletal pain . EmerginSo far, pediatric long COVID has been described globally by either researchers or family associations, although its incidence is still unclear since studies used different definitions of long COVID , designs , or populations  . In factNevertheless, as for other clinical problems related with SARS-CoV-2, the risk of developing long COVID in children may have changed during the pandemic. For example, severity of acute infection has changed with the surge of new variants and availability of vaccines , and risThe change in the risk of developing long COVID with different VOCs is unknown, and current uncertainties about the etiology of this condition make even more difficult to speculate about these differences. So far, a number of events have been associated with risk of long COVID, including SARS-CoV-2 persistence in different tissues , alteratGastrointestinal inflammation in pediatric COVID-19 has been recognized as an important aspect of the disease. While respiratory symptoms are more commonly associated with COVID-19, gastrointestinal symptoms have also been reported, particularly in children. Studies have shown that a significant number of pediatric COVID-19 cases present with gastrointestinal manifestations such as diarrhea, vomiting, and abdominal pain , 39.A retrospective cohort study of 12,306 pediatric COVID-19 patients in the United States . GastroiFurther research is needed to understand the precise mechanisms and long-term implications of gastrointestinal inflammation in pediatric COVID-19. By gaining a better understanding of this aspect of the disease, healthcare providers can improve diagnostic accuracy, management strategies, and patient outcomes in affected children.COVID-19 vaccines are generally safe and although the benefits of COVID-19 vaccination largely outweigh the risks, rare serious Adverse Events Following Immunizations (AEFIs) have been reported , 43. ApaAlthough most cases have a favorable outcome with a self-limiting course, recent data demonstrate that relapsing myocarditis episodes are also possible . We receGenetic variations among individuals may influence their susceptibility to developing severe inflammation in response to SARS-CoV-2 infection. Several studies in adults have identified genetic factors associated with an increased risk of severe COVID-19. Among these, the strongest and most robust finding for severity is represented by the 3p21.31 locus. However, few genetic insights are evident for children. Understanding these genetic predispositions can help identify individuals at higher risk and potentially inform targeted treatment strategies. Lee et al.  report tAcute and post-acute Inflammatory syndromes associated with SARS-CoV-2 infection represent a significant clinical challenge. It is worth noting that research on the relationship between inflammatory syndromes and SARS-CoV-2 is rapidly evolving. New studies and findings continue to emerge, providing further insights into the underlying mechanisms and potential therapeutic strategies. Recent insights into the pathogenesis of these syndromes have initiated to shed light on the complex and long-lasting interplay between the virus, the immune system, and systemic inflammation, opening new scenarios for a better characterization not only of SARS-CoV-2 associated conditions, but also other common pediatric pathologies like Kawasaki diseases and post-viral chronic fatigue syndromes. Understanding the underlying mechanisms and identifying therapeutic targets are crucial for developing effective preventive and management strategies. Future research should focus on unraveling the pathogenesis and the long-term consequences of these inflammatory syndromes and optimizing interventions to improve outcomes for affected individuals.


As the repercussions from the COVID-19 pandemic continue to unfold, an ever-expanding body of evidence suggests that infection also elicits pathophysiological manifestations within the central nervous system (CNS), known as neurological symptoms of post-acute sequelae of COVID infection (NeuroPASC). Although the neurological impairments and repercussions associated with NeuroPASC have been well described in the literature, its etiology remains to be fully characterized.This mini-review explores the current literature that elucidates various mechanisms underlining NeuroPASC, its players, and regulators, leading to persistent neuroinflammation of affected individuals. Specifically, we provide some insights into the various roles played by microglial and astroglial cell reactivity in NeuroPASC and how these cell subsets potentially contribute to neurological impairment in response to the direct or indirect mechanisms of CNS injury.A better understanding of the mechanisms and biomarkers associated with this maladaptive neuroimmune response will thus provide better diagnostic strategies for NeuroPASC and reveal new potential mechanisms for therapeutic intervention. Altogether, the elucidation of NeuroPASC pathogenesis will improve patient outcomes and mitigate the socioeconomic burden of this syndrome.  It has been established that the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entails long-term symptomatic repercussions in infected patients. The Center for Disease Control and Prevention (CDC) defines post-acute sequelae of COVID-19 (PASC) as the persistence of COVID-19 symptoms beyond 4 weeks of the initial infection  has been a growing area of concern, with its consequences referred to as post-acute neurological symptoms of COVID-19 (NeuroPASC). NeuroPASC's most prevalent symptom, cognitive impairment, has been reported in 28.85% of patients following COVID-19 infection according to a recent systematic review and meta-analysis . However solving . Various solving . Nonethe solving , 9, 11.  solving . Similar solving . Efforts solving . While c solving , 16, oth solving , 17. Non solving , 19, old solving , and pre solving  as risk Several theories have been proposed to explain these neurocognitive symptoms, including inflammatory changes, hypoxia, coagulopathy, vascular endothelial, dysfunction and direct viral invasion of the neurological tissue . AlthougMaintenance of optimal cognitive function is a complex process that requires coordination between neuron function and glial cells . In receComplex microglial–astrocyte interactions also form a delicate equilibrium in CNS health. Indeed, cellular dysfunction from either cell population or the maladaptive synergistic interactions between microglia and astrocytes can result in neurotoxicity and alter synaptic plasticity through numerous mechanisms , 30. WitUpon cerebral insult, astrocytes undergo drastic phenotype change referred to as reactive astrocytosis, induced due to an upregulation of pro-inflammatory cytokines by neuroinflammatory microglia such as (interleukin) IL-1α, IL-1β, tumor necrosis factor-alpha (TNF-α), and the complement component 1q (C1q) , 30, 33.As for oligodendrocytes, they are responsible for axonal myelination, which regulates action potential conduction velocity, essential for neural circuit dynamics . OligodeAltogether, the coordinated signaling between microglia, astrocytes, and oligodendrocytes is essential for homeostasis and CNS health.Acute and chronic CNS inflammation alike have drastic repercussions on glial circuitry and cytokine expression profiles, which result in dysfunctional immune signaling and synaptic plasticity . As a revia NF-κB signaling induces reactive astrocytosis, which in turn leads to excitotoxicity, white matter damage, and loss of myelin plasticity, in addition to oligodendrocyte and neuronal cell death (Neuroinflammation underlies one of the leading theories to explain CNS injury during SARS-CoV-2 infection and is a consequence of the well-documented systemic cytokine storm and subsequent increase in blood–brain barrier (BBB) permeability , 42. Thrll death , 35, 44.Neuroinflammatory pathways that alter CNS homeostasis are linked to cognitive and neuropsychiatric complications . The sysNeuroinflammation during acute SARS-CoV-2 infection may consequently induce brain parenchyma and vessel alterations that further foster the inflammation of neurons and supportive cells . Additioin vitro invasion assays on microglial cells co-cultured with SARS-CoV-2 demonstrate the loss of post-synaptic termini and neuronal cell death. Transcriptomic profiling of microglia exposed to SARS-CoV-2 revealed gene expression signatures that closely resembled neurodegenerative disorders  infiltration within the CNS and an abundance of activated microglia in the proximity of the infected tissue . The utiisorders . Neverthisorders , 54 whilisorders . Heterogvia the olfactory route . In a study utilizing human-pluripotent-stem-cell-derived brain organoids to examine SARS-CoV-2 neurotropism, ACE-2 positive choroid plexus epithelial cells were amenable to infection, which leads to an initial disruption of the blood–CSF barrier followed by a subsequent complete breakdown of barrier integrity . InfectiA recent study on AAV-hACE2 mice models with mild SARS-CoV-2 respiratory infection has demonstrated a prominent increase in pro-inflammatory cytokine and chemokine profiles  in the CSF and serum samples as rapid as 7 days post-infection . LongituCognitive dysfunction is also correlated with increased immunoregulatory pathway protein expression and a downregulation of inflammatory and antiviral response proteins . MoreoveConsidering the detrimental role of persistent microglial reactivity in neurodegenerative diseases, such reactive states could also be key to NeuroPASC pathogenesis. Accordingly, a key concept in AD trajectory known as microglial priming is associated with aging and systemic inflammation . FundameThere are considerable parallels between SARS-CoV-2 and influenza sequelae. Iosifescu et al.  and TaquAlternatively, microglial activation during acute SARS-CoV-2 infection could be sufficient to induce maladaptive inflammatory pathways, leading to chronic neuroinflammation and NeuroPASC in the absence of longitudinal peripheral stimuli. This phenomenon has been described following traumatic brain injury (TBI) in human brain samples, where densely packed reactive microglia are responsible for chronic neuroinflammation and white matter degradation . In factA comparable syndrome is cancer-therapy-related cognitive impairment, commonly referred to as “chemo-fog,” which is characterized by mild-to-moderate impairments in memory, attention, executive functioning, and processing speed . The terGlobally, the resolution of neuroinflammation is essential to mitigate neurological damage. Accordingly, this is the precise role of microglia and astrocytes subsets with tissue repair and anti-inflammatory functions , 92. HowThis mini-review has explored numerous cellular processes and pathways by which SARS-CoV-2 affects the CNS leading to glial reactivity and NeuroPASC . We illuReactive microglia are responsible for a plethora of CNS repercussions, including synaptic plasticity impairment , 95, inaWhile controversy surrounds the legitimacy of NeuroPASC as a distinct neuroinflammatory syndrome, evidence suggests that it possesses distinct microglial subtypes , humoralA thorough understanding of NeuroPASC pathophysiology and microglial reactivity is primordial to the development of disease-altering therapy. It is the first step toward alleviating the important socioeconomic burden of post-acute COVID-19 syndrome and its neurocognitive sequelae, a global health problem .JS: literature review, original and final manuscript redaction, and review. DC: original manuscript review. GR: final manuscript review and redaction. LC-W: original and final manuscript review. All authors contributed to the article and approved the submitted version.


Hepatic sinusoidal obstruction syndrome (HSOS) is caused by toxic injury, such as pyrrolizidine alkaloids, to the liver sinusoidal endothelial cells, and the gut microbiota may be involved. However, the specific role and underlying mechanism of gut microbiota in HSOS is unknown.HSOS model was established by gavage of monocrotaline (MCT) in rats. Fecal microbiota transplantation (FMT) with HSOS-derived or healthy gut flora was also conducted to validate the role of gut microflora in MCT-induced liver injury. The microbial 16 s rRNA analysis and untargeted metabolomics analysis in the faeces were performed to identify HSOS-related flora and metabolites. Finally, by supplementation with specific tryptophan metabolites, such as indole-3-acetaldehyde (IAAld) and indole acetic acid (IAA), we further confirmed the role of tryptophan metabolism in HSOS and the role of the AhR/Nrf2 pathway in MCT-induced liver injury.Bacteroides, Bifidobacterium, Lactobacillus and Clostridium, and accompanied by a decrease in microbial tryptophan metabolic activity and a series of tryptophan derivatives. Restoring the gut microbiota via FMT improved MCT-induced liver damage, while HSOS-derived gut microbiota aggravated the liver injury induced by MCT. Supplementation with microbial tryptophan derivatives (IAAld or IAA), or 6-formylindolocarbazole  could activate the AhR/Nrf2 signaling pathway, thereby attenuating the MCT-induced liver oxidative stress and liver sinusoidal endothelial cells injury.MCT induced HSOS-like liver injury in rats with significantly altered gut microbiota. Particularly, some tryptophan-metabolizing bacteria reduced in MCT-treated rats, such as Gut microbiota plays a critical role in MCT-induced HSOS, with inadequate microbial tryptophan metabolism in the gut and consequently a lower activity of the AhR/Nrf2 signaling pathway in the liver, which should be a potential target for the management of HSOS.The online version contains supplementary material available at 10.1186/s13578-023-01078-4. Hepatic sinusoidal obstruction syndrome (HSOS), also known as hepatic veno-occlusive disease (HVOD), is rare but life-threatening vascular liver disease with typical performance of painful hepatomegaly, ascites and jaundice , 2.gynura segetum (Tusanqi) which is a traditional Chinese herbal medicine rich in PAs carbazole.Additional file 2: Table S1. List of Primers for Real-time PCR.


Vaccines is a platform where scientists from different countries can present the current state of knowledge and the results of their studies in this area. Eleven interesting articles, including two review papers, were published there.The outbreak of the COVID-19 pandemic at the turn of 2019 and 2020 posed a substantial challenge for the world. The number of infected people and deaths reached unprecedented levels, paralyzing health services in many countries . RegardlIn a fascinating overview, Kaminska et al. (Contribution 1) presented the current knowledge on immunity after SARS-CoV-2 infection and vaccination against COVID-19. The authors also discussed the diagnostic and research tools available to examine the anti-SARS-CoV-2 cellular and humoral immune responses. In general, the immune responses generated after SARS-CoV-2 infection or vaccination seem similar. However, they differ in the details, e.g., higher post-vaccination antibody titers and a somewhat longer duration of post-infectious response. In addition, infection induces immune responses to a broader array of viral antigens. SARS-CoV-2 induces both humoral and cellular immune responses against spike (S), membrane (M), and nucleocapsid (N) antigens . ClinicaThe adaptive immune response consists of two complementary branches: humoral and cellular T cell-mediated immunity. Cellular immunity is less understood, largely due to a lack of validated diagnostic tests. T cells, with their diverse set of receptors, seem to have the advantage of recognizing a wider range of epitopes displayed in infected or antigen-presenting cells as being either an MHC class I or II surface protein. In the context of SARS-CoV-2 infection, while antibodies can protect us from developing an infection, T lymphocytes prevent the disease from becoming severe by eliminating infected cells . PrimoraTwo other papers are devoted to hybrid immunity against COVID-19, obtained via vaccination and SARS-CoV-2 infection at any order (Contributions 3 and 4). Nicola Serra et al. retrospectively evaluated antibody responses in a sample of 538 healthcare workers with a documented complete immunization cycle of three doses of mRNA vaccine against COVID-19 via multiplex assays. They found that patients who had also a history of asymptomatic SARS-CoV-2 infection had the highest titers of the anti-RBD and anti-S1 antibodies (Contribution 3). Blaszczuk et al. (Contribution 5) reported that individual SARS-CoV-2 variants can induce an immune response of varying strength in patients previously vaccinated against COVID-19, specifically the level of antibodies after SARS CoV-2 Omicron variant infection was lower than after Delta infection. A history of COVID-19 is associated with much stronger humoral immunity observed after vaccination in immunocompromised individuals . Hybrid COVID-19 is a threat to immunocompromised individuals, owing to their impaired natural immunity and suboptimal response to vaccines or immunosuppressive treatment. Consequently, they are at increased risk of hospitalization and death. These include subjects with primary immunodeficiency, end-stage renal disease patients, solid organ transplants, and subjects with solid tumors, among others . For insAlthough many aspects of COVID-19 immunity and vaccine response have already been clarified, there is still a sizable gap between clinicians’ questions and the available explanations, especially given the evolution of SARS-CoV-2 and new emerging variants. This will certainly be a subject of further research.


IL-33 and TSLP genes. The association between these genetic variations, cytokine production, and COVID-19 severity was examined. Results: Our findings revealed elevated levels of IL-33 and IL-25 in COVID-19-positive patients compared to COVID-19-negative patients (p < 0.05), indicating their potential as therapeutic targets for disease modulation. Moreover, a minor allele within the IL-33 gene (rs3939286) was found to be associated with a protective effect against severe COVID-19 (p < 0.05), and minor alleles of the TSLP gene (rs2289276 and rs13806933) were found to significantly reduce TSLP protein levels in serum (p < 0.05). Sex-specific effects of TSLP and IL-33 SNPs were observed, suggesting a potential influence of sex hormones and genetic variations on the regulation of cytokine production. Conclusion: The present study highlights the importance of alarmin cytokines and genetic variations in COVID-19 severity, providing valuable insights into personalized treatment approaches. Our results suggest that targeting alarmin cytokines may offer potential therapeutic benefits in managing COVID-19. Furthermore, the sex-specific effects of genetic variations emphasize the need to consider individual genetic profiles and sex differences when designing targeted interventions.Background: The immune response in COVID-19 is characterized by the release of alarmin cytokines, which play crucial roles in immune activation and inflammation. The interplay between these cytokines and genetic variations may influence disease severity and outcomes, while sex differences might further contribute to variations in the immune response. Methods: We measured the levels of alarmin cytokines in a cohort of COVID-19 and non-COVID-19 patients using a sensitive Meso Scale Discovery system. Additionally, we conducted an SNP analysis to identify genetic variations within the  COVID-19 (coronavirus disease 2019) is a respiratory disease caused by the SARS-CoV-2 virus and has a wide range of symptoms and complications. Extensive research has revealed the complex interplay of immune cell alterations, cytokine dysregulation, and genetic predisposition that play a pivotal role in the disease’s pathology [The dysregulation of cytokines is a crucial factor in the progression and severity of COVID-19. When the virus invades host cells, immune cells of the innate immune system including neutrophils, macrophages, eosinophils, and dendritic cells (DCs) migrate to the infected site and release proinflammatory cytokines such as interferons (INF), TNF (tumor necrosis factor), IL-6, IL-1, and chemokines CCL2, CCL3, and CCL4, which consequently leads to the recruitment of adaptive immune cells T cells to the inflamed area [An exaggerated and aberrant immune response triggers the excessive production of numerous inflammatory cytokines, creating a positive feedback loop. This cascade leads to the recruitment of additional immune cells and subsequent further cytokine production, resulting in widespread inflammation and potential organ damage . This phAmong the different cytokines produced in response to the viral infection, epithelium derived cytokines also known as alarmin cytokines including IL-33, TSLP , and IL-25, are among the first proteins released from the damaged host epithelial cells and play regulatory roles in both innate and adaptive immune responses. Alarmin cytokines initiate a series of events, triggering the migration of mast cells, DCs, and T cells to the inflamed regions of the lungs, where they work together to combat the virus. Detecting these cytokines in human samples has been challenging due to their low levels, making them difficult to detect with standard commercial assays. Advanced and sensitive detection methods are needed to accurately measure these cytokines and gain a better understanding of their role in immune responses and diseases like COVID-19 ,7.Clinical manifestations of COVID-19 exhibit considerable variability among individuals, which can be attributed to various factors, including genetics. Genetic variations can play a role in determining the extent and nature of the immune response mounted by each patient, thereby contributing to the diversity observed in COVID-19 severity and outcomes . Single TSLP are associated with the risk of asthma [TSLP and other alarmins on protein production and the severity of COVID-19 in affected patients, and to determine if alteration in the production of alarmins can serve as potential biomarkers for this viral respiratory infection.Previous studies have found that variations in the gene of the alarmin cytokine f asthma ,11,12,13The study was conducted following the approval of the Hamilton Integrated Research Ethics Committee. Between February and October 2021, COVID-19 patients as confirmed positive by real-time reverse-transcription-PCR (RT-PCR) were recruited upon hospital admission. Additionally, age- and sex-matched COVID-19-negative patients who were hospitalized but did not exhibit respiratory infections or respiratory tract-related diseases were included in the study as a control group. Demographic information, clinical characteristics, and outcomes of the patients were extracted from the electronic patient records. We adopted a categorization method previously described by the food and drug administration  to develIn COVID-19-positive patients, blood samples were obtained within 10 days of testing positive. Blood was collected into EDTA-containing vacutainers. Serum was evaluated for TSLP, IL-33, and IL-25 cytokine levels using an ultra-sensitive human cytokine multi-plex electrochemiluminescence-based immunoassay . The assay was performed using the MESO QuickPlex SQ 120 MM instrument  following the manufacturer’s protocol. The protein levels were quantified in picograms per milliliter of blood (pg/mL).TSLP gene  and IL-33 gene (rs3939286 and rs1342326), the TaqMan® SNP genotyping assay kit  was utilized. The assay was carried out in a 25 μL reaction volume, containing 12.50 μL of TaqMan genotyping master mix from Thermo Fisher Scientific, Waltham, MA, USA, 1.25 μL TaqMan genotyping assay mix (20×), and 11.25 μL DNA template. The thermal cycling conditions included an initial enzyme activation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The genotyping data were tested for Hardy–Weinberg equilibrium using Pearson’s chi-square test.Genomic DNA isolation was performed using the Qiagen DNAeasy extraction kit, following the manufacturer’s protocol for whole blood samples. DNA purity and concentration were measured using a Nanodrop spectrophotometer and diluted to a concentration of 10 ng/μL for optimal genotyping results. For genotyping analysis of single nucleotide polymorphisms (SNPs) in the t-tests and analysis of variance (ANOVA) and their non-parametric equivalents for data not normally distributed were performed to assess differences between groups. To investigate associations between individual polymorphisms and COVID-19 severity, multinomial regression analysis was employed, and odds ratios (OR) along with their 95% confidence intervals (CI) are reported.The data are presented as mean ± standard deviation (SD). A significance level (α) of 0.05 was used for statistical analysis. Data analysis was conducted using IBM SPSS Advanced Statistics  and GraphPad Prism software version 9.0 . Independent p < 0.05). Among patients, the proportion of non-smokers was significantly greater in the COVID-19-positive group in comparison to the COVID-19-negative group (p < 0.05).In total, 84 COVID-19-positive and 65 COVID-19-negative age- and sex-matched patients were recruited . Of the p = 0.635).The concentration of alarmin cytokines IL-33, TSLP, and IL-25 were compared in COVID-19-positive and -negative patients, and the association of alarmin cytokine levels with disease severity and age-related variations was evaluated . Signifip < 0.05), indicating an age-related variation in this cytokine  and IL-33 (rs3939286 and rs1342326). The frequency of homozygous major, heterozygous, and homozygous minor genotypes for these SNPs was not different when comparing the COVID-19-positive to COVID-19-negative patients  with an odds ratio of 0.109  indicating an inverse association. These results suggest that individuals carrying the minor genotype of rs3939286 have a protective effect against severe COVID-19 outcomes. In practical terms, this indicates that the presence of the minor genotype of rs3939286 may offer some level of protection to COVID-19-positive patients, reducing the likelihood of developing severe forms of the disease. The protective effect of the minor allele (T) of rs3939286 was consistently seen in all carriers (CT + TT), showing a strong association with reduced severity of COVID-19 .We conducted a multinomial regression analysis to explore potential relationships between severity . Among tTSLP and IL-33 SNPs revealed significant relationships between certain genetic variants and the production of TSLP and IL-33 proteins  demonstrating the mutant genotype leads to reduced protein levels. Further analysis based on gender showed sex-specific effects. Compared to non-carriers, males with the rs2289276 mutant allele and females with the rs3806933 mutant allele had significantly lower TSLP levels . Among the IL-33 SNPs, male carriers of the rs1342326 minor allele displayed significantly lower IL-33 levels compared to non-carriers (p = 0.0443), while no such difference was seen in females (p = 0.4212). Following the initial analysis, we conducted further tests to investigate whether the protective effects of these genotypes would be evident across different severities of the disease; however, we did not observe any significant relationship. One plausible reason for this lack of significance could be attributed to lower sample numbers within each severity category. These findings indicate that certain genetic variants in TSLP and IL-33 may influence protein production differently in males and females, suggesting potential sex-specific effects on disease outcomes.Regression analysis of proteins a. CarrieIn this study, we observed a higher prevalence of non-smokers among COVID-19-positive patients in contrast to COVID-19-negative patients. Similar findings have been documented in other observational studies . AlthougWe also observed significantly elevated levels of IL-33 and IL-25 proteins in COVID-19 patients compared to non-COVID individuals. These alarmin cytokines, released from damaged epithelial cells, play a crucial role in signaling danger to the immune system during viral infections. Interestingly, immune cells at inflamed sites also contributed to the production of these cytokines, suggesting a broader involvement beyond the epithelium . The senMaintaining a delicate balance of cytokine responses is vital for an effective immune defense against pathogens. Overproduction of alarmin cytokines may lead to dysregulated immune responses and exacerbate the pathophysiological consequences of the infection. Studies have indicated that blocking IL-25 can enhance antiviral immune activity . Additiop > 0.05). These findings suggest a potential effect of a longer steroid treatment period on IL-25 levels, indicating steroid treatment can modulate cytokine responses in COVID-19 patients.IL-33, TSLP, and IL-25 have been implicated as potential predictors of severe COVID-19 outcomes, associated with severe lung inflammation, making them promising candidates for disease severity control . HoweverIL-33, we discovered a significant inverse association between IL-33 SNP rs3939286 and COVID-19 severity, which meant that carriers of the minor genotype were found to be protected from developing more severe forms of COVID-19. While the role of IL-33 SNPs in viral infections, particularly COVID-19, remains largely unexplored, previous studies have indicated a protective effect of this SNP in other diseases, such as rheumatoid arthritis and cardiovascular conditions [The downstream pathways of IL-33 and TSLP have been shown to facilitate the recruitment of various immune cells and induce inflammation . In lighnditions ,30. It iTSLP SNPs, namely rs2289276, rs3806933, and rs1837253, in the general population is approximately 29%, 42%, and 26%, respectively. Furthermore, for IL-33 SNPs, the prevalence of rs1342326 and rs3939286 stands at 21% and 31%, respectively. These prevalence figures underscore the dynamic nature of genetic diversity across different genomic loci and populations [The prevalence of SNPs in the general population can vary depending on the specific SNP being considered and the demographic characteristics of the population studied. Different databases with large sample sizes such as α and TOPMED provide valuable resources regarding the prevalence of different SNPs in diverse populations. Data from these sources indicate that the prevalence of IL-33 SNPs on disease severity, we proceeded to explore the potential association of IL-33 and TSLP SNPs with protein production. Our analysis revealed that individuals carrying mutant alleles (CT + TT) of TSLP SNPs rs2289276 and rs3806033 exhibited significantly lower levels of serum TSLP. However, this reduced level was not observed in the case of rs1837253. This decrease in TSLP levels among minor genotypes could potentially influence disease severity and may offer protection against more severe outcomes. Nevertheless, our analysis did not establish a direct correlation, which could be attributed to the smaller sample size within each severity group.Following our investigation into the effects of TSLP gene’s promoter region. This region determines the fate of transcription and subsequent protein production. It is reasonable to speculate that the position of these SNPs on the promoter might play a regulatory role in TSLP protein production [TSLP SNPs on the production of each isoform remains an unexplored area in our study.The plausible explanation for the lower TSLP levels in these specific SNPs lies in their location on the oduction ,33. Addioduction . TherefoTSLP and IL-33 SNPs on protein production were sex specific. Specifically, in males, carriers of the rs2289276 mutant allele had significantly lower TSLP levels compared to non-carriers. Similarly, in females, carriers of the rs3806933 mutant allele exhibited lower TSLP levels compared to non-carriers. However, we did not observe significant differences in TSLP levels for the rs1837253 SNP in either sex. Additionally, among the IL-33 SNPs, we found a significant association between the rs1342326 minor allele and lower IL-33 levels in males, while no such association was observed in females. These sex-specific findings indicate that the genetic influence on alarmin cytokine production is different between males and females. The sex-specific pattern of these SNPs has been previously reported in other diseases [After stratifying our analysis based on sex, we found that the effects of the identified diseases ,12,35. Tdiseases .IL-33 and TSLP and COVID-19 outcomes. The identification of a significant inverse association between IL-33 SNP rs3939286 and COVID-19 severity suggests a potential genetic influence on disease outcomes, supporting the emerging evidence that human genetics may play a pivotal role in governing the clinical manifestations of SARS-CoV-2 infection which may have implications for risk stratification and personalized treatment approaches.This study provides novel insights into the role of alarmin cytokines, specifically IL-33, IL-25, and TSLP, in COVID-19. By utilizing a sensitive and advanced Meso Scale Discovery system, we were able to detect and quantify these cytokines accurately, despite their low levels in blood, offering valuable data for understanding the immune response in COVID-19 patients. We explored the potential associations between genetic polymorphisms in The limitations in this study include the relatively small sample size, which may have impacted the statistical power and ability to detect significant differences in some analyses. A larger cohort with diverse demographic characteristics could provide more robust and generalizable findings. While we observed associations between SNPs and cytokine levels, the study lacks functional data to elucidate the mechanisms driving these genetic influences. Further functional studies are needed to understand the underlying biology and pathways affected by these genetic variations. Future investigations could focus on developing targeted therapies that specifically regulate alarmin cytokine levels to mitigate the inflammatory response in severe cases. The sex-specific effects observed in this study underscore the importance of considering gender differences in disease pathogenesis and treatment. Integrating genetic information and sex-specific analyses could pave the way for personalized medicine approaches, tailoring treatments based on individual genetic profiles and sex-related factors.Overall, our findings suggest that alarmin cytokines  could serve as potential therapeutic targets to modulate disease severity in COVID-19, and genetic variations influencing protein production can also protect against developing severe COVID-19.


Lymphocytic interstitial pneumonia (LIP) is a benign lung disorder characterized by diffuse lymphocytic infiltration and polyclonal parenchymal proliferationA 52-year-old man was admitted to the internal medicine department because of weight loss. Serological testing revealed that he was HIV positive. Thoracic computed tomography (CT) was performed because he reported having flu-like symptoms for one month. Ground-glass opacities, accompanied by millimeter-sized thin-walled cysts, were observed in both lungs, which were more prominent in the upper lobes . Mediast-Approximately 5% of LIP cases undergo malignant transformation to lymphoma


OCT4, SOX2, KLF4 and MYC (OSKM) transforms differentiated cells into induced pluripotent stem cells. To refine our mechanistic understanding of reprogramming, especially during the early stages, we profiled chromatin accessibility and gene expression at single-cell resolution across a densely sampled time course of human fibroblast reprogramming. Using neural networks that map DNA sequence to ATAC-seq profiles at base-resolution, we annotated cell-state-specific predictive transcription factor (TF) motif syntax in regulatory elements, inferred affinity- and concentration-dependent dynamics of Tn5-bias corrected TF footprints, linked peaks to putative target genes, and elucidated rewiring of TF-to-gene cis-regulatory networks. Our models reveal that early in reprogramming, OSK, at supraphysiological concentrations, rapidly open transient regulatory elements by occupying non-canonical low-affinity binding sites. As OSK concentration falls, the accessibility of these transient elements decays as a function of motif affinity. We find that these OSK-dependent transient elements sequester the somatic TF AP-1. This redistribution is strongly associated with the silencing of fibroblast-specific genes within individual nuclei. Together, our integrated single-cell resource and models reveal insights into the cis-regulatory code of reprogramming at unprecedented resolution, connect TF stoichiometry and motif syntax to diversification of cell fate trajectories, and provide new perspectives on the dynamics and role of transient regulatory elements in somatic silencing.Ectopic expression of  POU5F1 (OCT4), SOX2, KLF4 and MYC (OSKM) in differentiated cells can reprogram them into induced pluripotent stem cells (iPSCs) (Ectopic expression of the Yamanaka transcription factors (TFs)  (iPSCs) . SimilarPioneering studies characterizing molecular features of fibroblast reprogramming have shown that overexpression of OSKM in somatic cells leads to extensive opening of closed chromatin, initiating multiple trajectories of cell state transitions, some of which culminate in successfully reprogrammed iPSCs  that accurately predict cell-state-specific ATAC-seq profiles at base-resolution from the underlying DNA sequence. We systematically annotated cell-state-specific predictive TF motif syntax in all regulatory elements, inferred dynamics of Tn5-bias corrected TF footprints as a function of TF concentration and motif affinity, linked dynamic elements to putative target genes, and elucidated rewiring of TF-to-gene cis-regulatory networks across cell state transitions in reprogramming. Our models reveal that early in reprogramming, OSK, at supraphysiological concentrations, rapidly open transient regulatory elements by occupying constellations of low-affinity binding sites, including non-canonical forms of the OCT-SOX heterodimer. The decay rate of accessibility of these transient elements is a function of motif affinity and OSK concentration. Additionally, these transient elements also contain occupied binding sites of somatic TFs such as AP-1. These binding sites are distinct from those found in fibroblast-specific regulatory elements and appear to sequester somatic TFs away from their fibroblast-specific sites. We identify a strong quantitative association between the sequestration of somatic TFs and silencing of fibroblast-specific genes, supporting a “repression-by-theft” hypothesis . We then harvested cells at D2, D4, D6, D8, D10, D12, and D14, as well as the starting population of fibroblasts (D0) and endpoint of iPSCs at passage 30, and processed them for scRNA-seq and scATAC-seq using the Chromium 10X platform  genes  and keratinocyte-like cells (T2), a primary reprogramming trajectory that terminates in iPSC cells (T3), and a parallel trajectory that ends in partially reprogrammed cells at day 14 (T4)  (HaghverS) genes  (MelchjoS) genes , and culpulation . Other fOTX2, TET1 and ZFP42 , which is likely due to MYC’s inability to directly initiate chromatin accessibility in contrast to the OSK pioneer TFs  based on single-cell expression levels of OSKM (MYC only. The population of keratinocyte-like cells (C6) along trajectory T2 expressed high levels of KM but not OS, reminiscent of a KLF4 driven keratinocyte-like off-target cell state found in scRNA-seq analysis of mouse reprogramming  can induce a keratinocyte-like fate   and partially reprogrammed (T4) trajectories. Cells that seemed to initiate reprogramming at day 2 express higher levels of all 4 factors, with  factors ,b. Post  factors . Median ~ 0 TPM) .While premature withdrawal of OSKM expression can stall the progress of cells in a partially reprogrammed state  and the In summary, we observe that cells with reprogramming potential express supraphysiological levels of all 4 reprogramming factors, many folds higher than in iPSCs, followed by a gradual reduction across the time course. Further, sustained expression of reprogramming factors at or beyond their levels in iPSCs may be critical to avoid partial reprogramming . ChromBPNet jointly models the total Tn5 insertion counts and their distribution (shape of the profiles) across these windows at single base resolution, as a function of the underlying sequence, while regressing out biases due to Tn5’s intrinsic sequence preference. We used a 10-fold, chromosome hold-out cross-validation scheme to train, tune, and evaluate the predictive performance of the models. We obtained high and stable Pearson correlation between total predicted and observed Tn5 insertion coverage in test regions across all folds and cell states (NANOG promoter (chr12:7788327-7790327) as it gains accessibility across the reprogramming time course  and retaining high contribution through the time course, including the iPSC state  and maintain accessibility throughout the time course post-induction. The KLF motifs once again show diminishing contributions over the time course with no contributions in the iPSC state, which is consistent with decreasing KLF4 expression across the time course and eventual loss in iPSCs . TFAP2 is predicted to transiently co-occupy accessible sites primarily with KLF in the Intermediate (C12) and Pre-iPSC states . NANOG expression is highest in the Pre-iPSC states  which correspond to extensive co-occupation of putative enhancers by OSK and TFAP2.We constructed cell-state-specific TF-to-gene (TF2G) networks regulating l states Methods.FN1 implicates somatic TFs AP-1, RUNX and TEAD as its primary regulators in fibroblasts. On induction of OSKM, the FN1 locus rapidly loses accessibility at multiple enhancers and by Day 4 its expression is reduced more than 10 fold  in the earliest Nanog enhancer (Enh1) resembles a partial version of known OCT-SOX motifs and scores poorly as a sequence match to any known motif from multiple motif databases . Hence, l states . We scanl states , MethodsNANOG locus, the uncorrected predicted scATAC-seq profiles closely match observed scATAC-seq profiles across all cell states and show a strong footprint over the CTCF motif  to the learned CTCF motif in a library of inaccessible GC-matched background sequences randomly sampled from the genome , top rowNext, to systematically study the influence of motif affinity and TF concentration on scATAC-seq footprints, we predicted a comprehensive constellation of marginal bias-corrected footprints for all 30 TF-MoDISco motifs over a range of sampled motif affinities binned into 3 strata , using ChromBPNet models from the 15 cell states which exhibit dramatic changes in TF concentrations across the reprogramming time course . The marKLF4 across cell states  with a marginal increase (~15 seconds) in the presence of Oct4 . HoweverThe somatic factor AP-1 also shows a strong relationship between marginal footprint strength, expression and motif affinity. The footprints are the strongest in fibroblasts and weakest in iPSCs. Weak affinity sites exhibit detectable footprints in fibroblasts which are nearly abolished in the xOSK state after OSKM induction . The in-These results demonstrate that ChromBPNet models trained on scATAC-seq data are able to reveal the intricate relationship between TF residence times, motif affinity and concentration on chromatin accessibility landscapes within and across cell states at unprecedented resolution.Overexpression of OSKM dramatically reconfigures the chromatin landscape of fibroblasts. For example, ~56% of the scATAC-seq peaks in the xOSK cells that first emerge 2 days post induction and anchor the primary reprogramming trajectory (T3) are not accessible in fibroblasts. An equivalent proportion of fibroblast peaks are closed in the xOSK state . PreviouWe first considered peak sets with distinct monotonic accessibility patterns over the course of reprogramming. The Open-Close peak set (OC1-4), consists of 148,901 fibroblast-specific peaks that close over the reprogramming time course. 8386 genes linked to OC peak sets are enriched for fibroblast terms including collagen formation and extracellular matrix assembly. OC peaks are predicted to be regulated by AP-1, ATF, CEBP, RUNX, IRF1, NFI, and FOX motifs.ZIC TFs  or late  during reprogramming. 8118 genes linked to Close-Early Open (CO/E) peaks are involved in stem cell regulation and RNA metabolic process, while 3860 genes linked to Close-Late Open (CO/L) peaks are enriched for cell-cell adhesion and neurogenesis processes, consistent with previous analyses  cells  are closed in fibroblasts but open at the first time point after OSKM induction in the xOSK (C8) state, before closing eventually in iPSCs. COC/E peaks are enriched primarily for OSK motifs, suggesting that they arise as an immediate consequence of OSKM overexpression. This reinforces the idea that OSK can act as pioneer factors and recognize their cognate motifs on nucleosomal DNA and open closed chromatin . In addi4) cells , the los4) cells  further 4) cells   are also closed in fibroblasts, but attain maximum accessibility among reprogramming cell states between days 4–14, and then close in iPSCs. COC/L peaks likely arise due to secondary effects of OSKM overexpression. In contrast to Early Transient peaks that primarily show OSK and AP-1 binding, Late Transient (COC/L) peaks are relatively depleted for O binding but displayed high scores for TFAP2 motifs . TFAP2C,gramming . COC/L pSC cells . This iske state . The hybKRT16 and KRT14, and enriched for the GO term cornification. As expected, Keratinocyte-like specific peaks (K1) are enriched primarily for KLF motifs but not OCT-SOX. Finally, constitutively accessible peaks  are localized near both fibroblast and iPSC genes, as well as housekeeping genes such as GAPDH and RPS11.Additionally, a set of keratinocyte-specific peaks  is highly accessible in the keratinocyte-like population (C6), linked with 781 genes including keratinocyte markers such as TFAP2 and ZIC, that at later stages of reprogramming cooperatively bind with OSK to reinforce the pluripotency circuit. Beyond peaks with monotonic accessibility patterns, nearly 40% of all dynamic peaks along the primary reprogramming trajectory (T3) are transient, and drive poorly characterized gene expression programs.Thus, overexpression of OSKM triggers a dramatic, multi-phasic remodeling of chromatin accessibility and gene expression involving 444,962 dynamic scATAC-seq peaks linked to 12,135 dynamic genes including 1074 TFs. Consistent with previous analyses, while some iPSC-specific peaks are gained early during reprogramming, the majority are acquired during the late stages of reprogramming (To decipher the role of the Early Transient peaks (COC/E), we first checked whether they overlapped accessible sites in a reference DNase I hypersensitive sites (DHS) index across 438 well characterized cell-lines, primary cells and tissues , MethodsTo determine whether these early transient peaks are a general feature of reprogramming, we compared the Early Transient peak sets (COC/E1-3) and a contrastive set of peaks (E-ON) that opened early at Day 2 and remained accessible throughout our time course against OSKM ChIP-seq  data froOCT4 and SOX2 are 4-fold and 8-fold higher in xOSK compared to iPSCs (C15). Further, OSK motifs disproportionately dominate the chromatin landscape in the xOSK state . OS expression is sustained over the course of reprogramming through iPSCs, and switches from exogenous to endogenous over the time course. However, the mRNA levels of SK state . These oWe suspected that supraphysiological concentration and stoichiometry of OS in the xOSK states could enable these pioneer factors to occupy low affinity, non-canonical binding sites resulting in esoteric landscapes of chromatin accessibility. Based on this hypothesis, we expected early transient peaks in the xOSK state to be enriched for non-canonical OCT-SOX binding sites.de novo OCT-SOX motifs inferred by TF-MoDISco from ChromBPNet models of xOSK and iPSC states to a canonical OCT-SOX motif (HD) and POU-specific (POUS) domains respectively, and a trailing ACAA subsequence that binds to the SOX2 HMG domain   , suggestS domain. Further, in OCT-SOX sub-motifs 2,3 and 5, the leading ATTT subsequence that binds the OCT4 POUHD domain is greatly attenuated, suggesting that a partial POUS-HMG heterodimer motif in transient peaks may be sufficient to recruit the OCT4-SOX2 heterodimer at high concentrations in the xOSK state. A recent study supports our hypothesis by showing that OCT4-SOX2 heterodimers can in fact bind nucleosome-embedded instances of this exact partial motif and drive chromatin accessibility . COC/E3 peaks exhibit the slowest decay and are accessible through intermediate cell state 12 (day 8) until the Pre-iPSC state (day 10). The variable decay rates across these peak subsets could be due to subtle differences in the affinities of non-canonical OSK motif instances in each of these subsets. To test this hypothesis, we compared log-odds scores of instances of the OCT-SOX, KLF and SOX motifs across the three subsets of early transient peaks. We further contrasted these early transient peak subsets against a control set of peaks (E-ON) that also gain accessibility at day 2 but remain accessible throughout the trajectory and in iPSCs , MethodsOCT4/SOX2 expression levels across all the cell states in the primary reprogramming trajectory. These surrogate binding saturation curves for OCT-SOX not only show that accessibility decreases with TF concentration but also that the decay rates are faster for peaks with lower affinity OCT-SOX motifs  at days 1 and 2 during the early stages of reprogramming , MethodsCOL1A1 and FN1 showed reduced expression as early as day 1  between the fibroblast gene program score and the AP-1 sequestration score over early transient peaks . Together, these results suggest that overexpression of OSKM has a quantitative effect on somatic silencing in the early stages of reprogramming, mediated by redistribution of somatic TFs to newly opened regulatory elements.We then used the multiome data to test for quantitative association between the degree of sequestration of AP-1 to early transient peaks and repression of fibroblast-specific genes. For each nucleus, we estimated an “AP-1 sequestration score” as the proportion of ATAC-seq reads falling in Early Transient peaks containing predictive AP-1 motif instances relative to total reads in all peaks . Focusinnt peaks . This nent peaks ,c. In coOur study provides a comprehensive understanding of somatic cell reprogramming by combining single-cell data with an analysis of the interplay between temporal TF stoichiometry and motif syntax. Our single-cell RNA-seq and ATAC-seq atlas is a high-resolution and temporally dense profiling of reprogramming intermediates, in contrast to past efforts that sorted reprogramming intermediates based on surface marker expression (de novo representations that highlight bases that quantitatively contribute to accessibility profiles without the need to specify arbitrary affinity thresholds. For a given TF, its relative contribution to accessibility drops with decreasing concentration, which is reflected both in the contribution scores and bias-corrected footprint shapes (Our work showcases the potential of using bias-corrected, base-pair resolution neural networks to unravel stoichiometry-dependent low-affinity TF binding. In contrast to classical motif analysis methods such as PWM scanning, which suffer from low specificity when detecting low affinity TF binding sites, ChromBPNet learns t shapes . This enOSKM combinatorially and quantitatively reconfigure the somatic cell state. Upon induction, stochastic variation in exogenous OSKM stoichiometry results in initial diversification into a putative reprogramming trajectory, and off-target keratinocyte-like and fibroblast-like fates. In cells with supraphysiological overexpression of all factors, OSK rapidly dominate the chromatin landscape by extensively binding to and opening closed chromatin, while fibroblast-specific peaks close early –iii. NewS-HMG binding motifs. These observations suggest that the initiation of reprogramming is not tightly coupled to OSK binding specificity. This is supported by reports which show that substituting OCT4 with mutant OCT4 or orthologous OCT6 in a reprogramming cocktail is able to initiate reprogramming in a manner similar to OCT4 (Within Early Transient regulatory elements, we find pervasive evidence of low-affinity binding sites of OSK, including an excess of partial POU to OCT4 , suggest to OCT4 . Late Cl to OCT4 . Such inSPI1 in T-cell development (Our study provides new insights into the role of Early Transient peaks in somatic silencing. The ChromBPNet models revealed that Early Transient peaks harbor thousands of instances of AP-1 motifs, which was supported by the presence of discernable footprints , 7ii. Thelopment . The dayelopment .Together, this study provides a template for studying the effects of heterogeneous stoichiometry on cis-regulation. We exploit the stochasticity of Sendai virus induction to study the combinatorial state space of TF overexpression in reprogramming, and leverage powerful neural network models that learn cell state, TF stoichiometry-aware mappings from DNA sequence to chromatin accessibility. Our approach outlines a powerful paradigm for interrogating the stoichiometric and cis-regulatory sequence determinants of factor induced de-differentiation and trans-differentiation trajectories.Our analysis is subject to certain limitations. First, the cell state transition trajectories identified in our study represent our best guess estimate by combining multiple lines of evidence including pseudotime inference, Sendai virus dilution, and findings from previous studies. Validating these trajectories would necessitate the use of lineage tracing methods. Second, the peak-gene linking is approximate, in part due to computational linking of scATAC-seq and scRNA-seq cells. This can impact the accuracy of the T2G networks. Third, ChromBPNet models explain chromatin dynamics that are predicted by local 2kb sequence windows. The models may be inaccurate when chromatin dynamics are determined by long-range interactions, and changes to methylation and histone modifications. For example, dynamic CTCF peaks that are closed in fibroblasts and open over the course of reprogramming are incorrectly predicted to be constitutively accessible by the models. To partially overcome this limitation, we restrict our analysis to peaks that are accessible in the respective cell types. Improvements in experimental, computational and modeling approaches will yield increasingly accurate and precise insights into the molecular mechanisms underlying cellular reprogramming. That said, our analytic approach should prove of broad utility and pave the way for new analyses and insights into nuclear reprogramming and TF behavior that may have previously been missed.Supplement 1Supplement 2Supplement 3Supplement 4Supplement 5


Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the expansion of immature myeloid cells in the bone marrow (BM) and peripheral blood (PB) resulting in failure of normal hematopoiesis and life-threating cytopenia. Allogeneic hematopoietic stem cell transplantation  is an established therapy with curative potential. Nevertheless, post-transplant relapse is common and associated with poor prognosis, representing the major cause of death after allo-HCT. The occurrence of relapse after initially successful allo-HCT indicates that the donor immune system is first able to control the leukemia, which at a later stage develops evasion strategies to escape from immune surveillance. In this review we first provide a comprehensive overview of current knowledge regarding immune escape in AML after allo-HCT, including dysregulated HLA, alterations in immune checkpoints and changes leading to an immunosuppressive tumor microenvironment. In the second part, we draw the line from bench to bedside and elucidate to what extend immune escape mechanisms of relapsed AML are yet exploited in treatment strategies. Finally, we give an outlook how new emerging technologies could help to improve the therapy for these patients, and elucidate potential new treatment options.The online version contains supplementary material available at 10.1186/s12943-023-01889-6. Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by clonal expansion of immature myeloid cells in the bone marrow (BM) and peripheral blood (PB), resulting in failure of normal hematopoiesis and life-threating cytopenia . For theRegarding treatment for relapse post allo-HCT, long-term remission is mainly achieved by treatment concepts that include any type of cellular immunotherapy, such as donor lymphocyte infusion (DLI) or a second allo-HCT  . NeverthIn this review, we summarize different ways of immune escape in AML patients after allo-HCT, separating mechanisms leading to impaired HLA expression from those characterized by aberrant immune checkpoint expression and differential effects leading to a remodeling of the TME. Beyond, we discuss implications for present and future therapeutic approaches and give an outlook on how new innovative technologies could help to identify new targets to overcome immune escape in relapsing AML.An intact antigen-presentation machinery is critical for an effective recognition of leukemia cells by the donor immune system. HLA possess outstanding ability to elicit an immune response either by presentation of variable peptides such as minor histocompatibility antigens (miHag) and tumor-associated antigens (TAA), or as direct target. The genomic HLA molecule disparity between donor and recipient triggers T-cell allo-recognition . In contImpaired HLA expression is a quite rare event in hematologic neoplasms at time of diagnosis, but frequently occurs in patients with relapse after allo-HCT –15. HencIn the setting of HLA-matched allo-HCT, non-genomic loss of HLA expression seems to play a major role for post-transplant relapse. Two seminal studies including patients transplanted from matched sibling donors (MSD), MUD, and MMUD  and haplThe molecular mechanism behind HLA dysfunction on leukemia cells has been extensively studied. Downregulation of MHC class II transactivator CIITA (MHC2TA) by hypermethylation of its promotor has been detected by gene expression analysis , 17. BeyWhereas the studies published so far mainly comprise HLA class II molecules, recent work reported HLA aberrations also including HLA class I, which were frequently detected in relapsing AML patients . Hence, + cells with subsequent DLI) [During the last decade, allo-HCT from haploidentical family donors (haplo-HCT) has become a frequently used transplant strategy for allo-HCT in patients lacking a HLA matched donor . In thisent DLI) . Lack ofent DLI) , 30. As ent DLI) , 31. It ent DLI)  as a mecent DLI) –34, withent DLI) , 36. Intent DLI) .The temporal and biological origin of HLA loss has not yet been completely elucidated. Although rare alterations involving HLA at the time of diagnosis in patients with hematological cancers were reported , 30, theApparently, HLA loss is not exclusive of haplo-HCT and AML, but was documented in AML relapses after matched unrelated (MUD) and mismatch unrelated donor (MMUD) allo-HCT , and alsThe understanding of immune checkpoints (ICP) has revolutionized the role of immunotherapy in cancer treatment. In healthy individuals ICP on immune effector cells represent physiological control mechanisms essential for maintaining immune tolerance and preventing autoimmunity .Programmed cell death protein 1 (PD-1) and its ligand PD-L1 constitute a major inhibitory axis. Upon engagement of these two proteins, T-cells exhibit a hyporesponsive T-cell differentiation state defined by poor effector function, sustained expression of inhibitory receptors, lack of response to stimuli and a transcriptional state distinct from that of functional effector or memory T-cells [regs). The resulting T-cell suppression was associated with AML relapse after allo-HCT [ T-cells . The PD- T-cells . An aber T-cells , 43. PD- T-cells  and simi T-cells , 45. Viaallo-HCT , 46–48.cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is an ICP molecule that binds to the ligands CD80 and CD86 in a competitive manner with CD28. Normally, CD80 and CD86 interact with CD28 to provide a co-stimulatory signal to T-cell receptor (TCR) mediated activation. In contrast, interactions of these ligands with CTLA-4 antagonize CD28-mediated co-stimulation and inhibit T-cell responses [reg cells or activated conventional T-cells due to active endocytosis from the plasma membrane, from where it can be recycled to the surface or degraded in lysosomal compartments [The inhibitory receptor esponses . The bioartments . CD80 anartments , 47, furartments .T-cell immunoglobulin and mucin domain 3 (TIM-3), which physiologically binds galectin-9 , is highly expressed on AML blasts, and plays a major role in immune escape of AML and relapse post allo-HCT [+ T-cells was strongly associated with T-cell exhaustion and relapse post allo-HCT, whereby PD-1/TIM-3 positive T-cells of PB were already detected 2 months before clinical relapse, suggesting a predictive value of this observation [+ T-cells from relapsing AML patients expressing TIM-3, PD-1 and CTLA-4, have been reported, while their corresponding ligands Gal-9, PDL-1, CD80, and CD86 were expressed on leukemic blasts [Apart from PD-1/PD-L1 and CTLA-4, allo-HCT . Interesallo-HCT . Gal-9 iallo-HCT , 53. Konervation . Additioc blasts .T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is another inhibitory receptor present on NK and T-cells. Ligands for TIGIT are the poliovirus receptors  and PVRL2 . TIGIT binds to these ligands in competition with the activating costimulatory receptor DNAM-1, thereby suppressing the activity of NK and T-cells [+ T-cells early after allo-HCT are associated with AML relapse [ T-cells , 57. Var T-cells , 58, 59. relapse . TIGIT,  relapse . Moreove relapse , 58, 62.KLRG-1 was reported to be involved in AML relapses post allo-HCT by Hutten et al.; importantly this study highlighted the simultaneous expression of multiple inhibitory checkpoints (PD-1/TIGIT/KLRG-1) on antigen specific CD8 T-cells of the PB as a key feature of T-cell dysfunction [Another checkpoint, function .CD47 represents a macrophage immune checkpoint highly expressed on leukemic stem cells (LSCs) and AML cells, leading to immune evasion through the inhibition of phagocytosis [ocytosis . In thisocytosis , whereatocytosis , 66, wheocytosis .CD200, which is a new putative checkpoint on LSCs in AML and was significantly overexpressed on these cells [Another surface molecule in this context is se cells . Positivse cells . A recense cells . CD200 ese cells  indicatiFurther inhibitory immune checkpoint receptors/ligands such as LAG-3, VISTA, 2B4, B7-H3, B7-H4, and LILRB4 [d LILRB4 , 72, andd LILRB4  may be iIn summary, the overexpression or upregulation of checkpoints on T-cells or the corresponding ligands on AML blasts has been reported in up to 40% of AML patients  who therThe tumor microenvironment (TME) comprises a complex network of immune cells and non-cellular components. Malignant cells are able to change various physiological processes within the TME to their advantage. Hence, both immune effector cell dysfunction and alterations of the non-cellular components may contribute to tumor immune escape by remodelling of the TME towards an immunosuppressive phenotype.Beyond T-cell dysfunction associated with HLA loss/downregulation and ICP, other mechanisms leading to impaired T-cell reactivity in AML have been described.The formation of an immunological synapse among T-cells and myeloid blasts is formed in a multistep process leading to adhesion between T-cell and antigen-presenting cell (APC) as well as T-cell and blasts. For this complex formation, the actin cytoskeleton and the polymerization of actin is critical . Le Dieucms,, was identified early after transplantation. This finding suggests limited and exhausted T-cell immunity early post HCT to be a strong risk factor for AML relapse and therefore part of the immune evasion process [Noviello et al. analysed the T-cell compartment of AML patients relapsing after allo-HCT and reported the presence of exhausted BM T-cells with restricted TCR repertoire and impaired T-cell effector functions, including reduced IL-2, γ-IFN and TNFα secretion and lower degranulation rates. Beyond, a small fraction of severely exhausted T process . Remarka process .regs) control the immune homeostasis via induction of immunosuppression [regs are highly enriched in the blood and/or BM of myeloid leukemias after allo-HCT [regs in patients and healthy controls. However, Tregs from AML patients showed an activated phenotype (CD45RA−HLA-DR+) indicating a potential role of these cells in relapse [regs are not able to suppress the GvL effect in AML, and suggest that the predominance of these Tregs could even be a favourable prognostic marker [regs as a kind of a double-edged sword, as they can have opposite effects promoting relapse and at the same time preventing the development of graft-versus-host disease (GvHD).Regulatory T-cells  , 107. NKFurther cell types potentially contributing to immune escape are tumor-associated-macrophages (TAMs). TAMs are characterized by anti-inflammatory activity  and haveHematologic tumors may alter the TME by switching cytokine production from pro-inflammatory to anti-inflammatory and by increasing the release of immunosuppressive molecules. As an example, the physiological function of IL-15 comprises the expansion and activation of effector T-cells and NK-cells and the promotion of memory T-cell generation. In the post-transplantation setting of different haematological malignancies low plasma levels of IL-15 have been associated with a higher risk of relapse . In AML,Apart from IL-15, further members of the cytokine network in the TME play a prominent role in the post-transplant setting, including the chemokine receptor CXCR4 and its ligand CXCL12. CXCR4 belongs to the group of transmembrane G-coupled protein receptors and is expressed on normal stem cells as well as AML blasts controlling the migration of LSCs to the BM . Increas+ T-cells from relapsing AML patients after allo-HCT, indicating an important role for this cytokine in immune evasion of AML [ex-vivo setting the impact of transforming growth factor beta 1 (TGF-β1), which induced NK-cell dysfunction in AML patients with early relapse after allo-HCT. This effect was reversible by pharmacologic inhibition of TGF-β1 signalling in leukemia xenograft mouse models [Studies about chemokine profiles prior and post BM transplantation as recently performed for CXCL1, CXCL10 and CXCL12 , might in of AML . Moreoven of AML . A recene models , indicate models , 133.regs [Closely linked to the cytokine milieu in the TME are metabolic active molecules that have been reported to modulate the TME in leukemia. One example is Arginase II, of which high plasma levels in AML patients, contributing to an immunosuppressive environment by blocking T-cell functions and inducing the suppressive M2-like phenotype of macrophages . Hence, regs . IDO1 exregs –138. Tworegs . In CD73regs , indicatRegarding other metabolic processes, glycolysis has been shown to play a critical role regarding immune evasion in AML: In patients relapsing after allo-HCT it was shown that in response to leukemia-derived lactic acid, the T-cells exhibited reduced glycolysis, functionally leading to decrease in proliferation and anti-leukemic activity. Sodium bicarbonate restored the metabolic fitness of donor T-cells both in vitro and in vivo after application to patients with AML relapse post-transplant receiving treatment with DLI .regs and Myeloid-derived suppressor cells (MDSCs) play a role, which both exert an immunosuppressive function by damping anti-tumor T-cell responses [regs of AML patients relapsing after allo-HCT [reg levels, MDSCs subsets were increased early post-transplantation, but decreased over time. Nevertheless, no association between MDSCs and Tregs with subsequent relapse could be found [Related to cytokines and enzymes are the secreting cellular components, which increase the immunosuppressive phenotype of AML. Among these, Tesponses . A recenallo-HCT  and showbe found .Tumor infiltrating lymphocytes (TILs) comprise a heterogenous cluster of cells closely located to the tumor to create an anti-tumor response . TILs haMesenchymal stroma cells (MSCs) have been shown to inhibit NK-cell-mediated killing of AML blasts and in general exhibit a more immunosuppressive phenotype than MSCs from healthy donors –148. PosFinally, the vascular endothelium plays a major role for AML progression, as remodelling of the vascular compartment has been shown including high vascular permeability and reduced blood flow impairing the distribution of drugs and immune cells . M2-macrA summary of immune escape mechanisms described after allo-HCT for AML is depicted in Fig. So far, no uniform standard treatment for AML relapse after allo-HCT has been defined, nor have approaches specifically addressing immune escape mechanisms become clinical routine. In principle, initial disease control is approached by either conventional chemotherapy, hypomethylation agents (HMA) ± venetoclax, or specific therapies targeting mutations (tyrosine kinases or IDH). However, subsequent cellular therapies relying on an allogeneic immune reaction – i.e. DLI or a second allogeneic HCT —are regarded mandatory. , 153. AlIncreasing knowledge on mechanisms of relapse have also allowed deeper insights into the interplay between the leukemia and the allogeneic immune system under currently used treatments. As an example, HMA have been shown to augment the GvL-effect by inducing cytotoxic CD8 + T-cell responses against tumor-associated antigens , 166, alSo far, outside of clinical trials, the immune escape mechanisms described in this review are not yet routinely considered neither for treatment choice nor incorporation of specific elements into the management of post-transplant relapse. In this section we summarize published clinical experience from therapeutic approaches considering particular immune escape mechanisms.in-vitro experiments testing the activity of CD123-directed immunotherapies  both in cell lines and xenograft models from patients with relapsed AML after allo-HCT with low baseline MHC-II expression [When HLA class II downregulation was described as a mechanism of immune escape of AML after allo-HCT, it was evident, that this phenomenon was of epigenetic nature and therefore potentially reversible. As discussed in the previous paragraph, induction of IFNγ release e.g. as a consequence of DLI-induced GvHD might restore the expression of HLA II on blasts. In that sense, DLI addresses immune escape by reversion of HLA downregulation, a finding that is thought to explain in part the correlation of GvHD with GvL effects. Similarly, significant increase in the expression of MHC-II molecules and killing of leukemia cells was found to be mediated by IFNγ secretion from CD4 T-cells in a series of pression . Hence, pression .In cases of HLA loss, the allogeneic effect mediated by T-cells is practically abrogated, as demonstrated by Vago et al. . DLI or Immune checkpoint blockade (ICB) has shown activity in some hematologic disorders, especially lymphoid malignancies . After aDavids et al. were among the first to explore the safety and efficacy of ICB after allo-HCT. In two prospective phase I trials they used ipilimumab  and nivoregs and CD4 cells was associated with response [+ T-cells and a decrease in exhausted CD8+ T-cells was observed.According to these findings, the risk/benefit ratio does not support the implementation of ICB for the general population with myeloid malignancies relapsing after allo-HCT. Hence, the identification of subgroups with a higher chance for response seems reasonable. Penter et al.  identifiresponse . Penter response  analysed+ T-cells in BM of patients responding to azacytidine/nivolumab, and high CTLA-4 expression on CD4+ T-cells in non-responders, whereas the baseline expression of PD-L1 on blasts/ PD-1 on CD3+ T-cells was not predictive for response. Penter et al. performed integrative transcriptome-based analyses after treatment with decitabine/ipilimumab [T-cells to blast ratio was a major determinant for responses, reinforcing the role of low disease burden for long-term remission to immunotherapy. In extramedullary AML, a high infiltration of regulatory Treg-cells and the CTLA-4 or PD-1 expression on tissue resident T-cells predicted the response to ipilimumab.Since upregulation of PD-L1 on leukemic cells might also be responsible for resistance to HMA treatment , ICB havilimumab . Among tBased on the role of IL-15 for the activation of immune effector cells and the observation of reduced plasma levels in patients relapsing after allo-HCT, recombinant IL-15 has been clinically studied in combination with NK-cell infusion. However, data were conflicting, since remission rates of 35% were observed in relapsed/refractory AML upon haploidentical NK-cell infusions plus IL-15 , whereasA summary of preclinical and clinical studies on treatment options addressing distinct immune escape mechanism are listed in Table in-vivo models became more and more popular. Advanced cell culture systems comprising 3D biomimetic scaffolds or organ-on-chip technologies may overcome the limitations of traditional cell culture models lacking TME cells or other external factors by mimicking the bone marrow environment. Hence, these assays provide optimized conditions for studying AML in the context of relapse and drug resistance [Despite the increasing number of studies about relapsing AML after allo-HCT, further investigation is needed to achieve a deeper, more comprehensive understanding of the immune mechanisms behind post-transplant relapse, allowing for rapid analysis and individualized treatment. Rapidly evolving techniques, including multiomics factor analysis , 18, 188sistance , 194.AML relapse after allo-HCT remains a challenging problem. It is hoped that the rapidly increasing knowledge of immune escape mechanisms might allow to exactly define the particular mechanism(s) responsible for AML relapse in an individual patient and hence develop a specific, pathophysiology-based and individualized treatment strategy. This is of particular relevance since at least some immune escape mechanisms, such as downregulation of HLA and increase of immune checkpoints, seem to appear in a mutually exclusive way , which iFor the time being, the clinical application of specific treatment options is still at the very beginning and has not made its way into clinical practice outside clinical trials. However, a broad variety of identified mechanisms seem to offer attractive opportunities for specific interventions. Among approaches to revert HLA downregulation, the application of IFNγ is investigated in a clinical trial (NCT04628338). Beyond, EZH2 inhibition appears particularly promising in this context. In contrast to the IFNγ-dependent strategies, re-establishing HLA expression via EZH2-inhibition was not associated with PDL-1 upregulation . ComparaAs discussed above, currently available ICB had only moderate effects against myeloid malignancies, which is why their use at least as monotherapy is unlikely to become a therapeutic option in the post-HCT setting. The combined role of several checkpoint ligands simultaneously expressed on blasts from the same patient , as wellWith respect to systemic application of cytokine-based therapies, clinical evidence is still extremely limited. Co-administration of IL-15 and immune effector cells clinical results have revealed conflicting data. Hence, although promising, refinement of current concepts is required for successful clinical use. An approach that is closer to clinical evaluation is the systemic application of sodium bicarbonate for metabolic reprogramming of allogeneic T-cells, which has been proven its feasibility in humans and may have the potential of reinforce the GvL effect by counteracting the AML-mediated metabolic and functional inhibition of allogeneic T-cells .https://clinicaltrials.gov/, upon August 8th, 2023.). Of note, about half of these trials investigate immune modulating principles as maintenance for prevention of relapse in high-risk patients, i.e. in a setting without highly proliferating leukemia, to allow more time for immune effects to develop. Beyond, also studies investigating different cellular therapy-based strategies are listed, whose detailed description is out of the scope of this review.Given the broad variety of therapeutic toeholds, there is an enormous number of ongoing clinical trial in the field. To provide an overview on the range of current activities, ongoing clinical trials targeting immune escape mechanisms in the post-transplant setting are summarized in the online supplement (see Additional file In conclusion, we are facing a fascinating era of increasing understanding of immune escape mechanisms that allow myeloid blasts to evade from the allogeneic control after allo-HCT. Whereas preclinical research will continue to elucidate further evasion mechanisms as well as how these different mechanisms may interact, ongoing and future clinical trials will have to identify those mechanisms that can be successfully addressed to provide rational and individualized treatment to patients with imminent or overt relapse after allo-HCT.Additional file 1:Additional Table 1. Ongoing clinical trials investigating pathomechanism-derived approaches post allo-HCT*.