The alteration of host cells Ca2+ homeostasis is one of the strategies that viruses use to modulate host cells signal transduction mechanisms in their favor. Host calcium-permeable channels and pumps  mediate Ca2+ across the plasma membrane or subcellular organelles, modulating intracellular free Ca2+. Therefore, these Ca2+ channels or pumps present important aspects of viral pathogenesis and virus–host interaction. It has been reported that viruses hijack host calcium channels or pumps, disturbing the cellular homeostatic balance of Ca2+. Such a disturbance benefits virus lifecycles while inducing host cells’ morbidity. Evidence has emerged that pharmacologically targeting the calcium channel or calcium release from the endoplasmic reticulum (ER) can obstruct virus lifecycles. Impeding virus-induced abnormal intracellular Ca2+ homeostasis is becoming a useful strategy in the development of potent antiviral drugs. In this present review, the recent identified cellular calcium channels and pumps as targets for virus attack are emphasized.Ca Viruses exploit the environment of host cells to replicate, thereby inducing host cells’ dysfunction. Virus–host interaction is the foundation of pathogenesis and closely associated with disease severity and incidence. The prevention and therapy of virus infections are often confounded by the high mutation rates that facilitate the viral evasion of antiviral strategies that target virally encoded proteins. Modulations of the intracellular environment have become an important strategy in antiviral drug discovery and development. In mammalian cells, Caormation ,2. It hantration . A virus2+ channels and pumps are distributed on the cell plasma membrane. Therefore, these membrane proteins become the direct target of virus infection. Interaction between viruses and these membrane proteins is the foremost approach of viruses perturbing the host cell calcium signal system. This interaction may inhibit or stimulate calcium influx and modulate free cytosolic Ca2+ concentrations. After entry into the host cell, viruses stimulate or inhibit the calcium release from internal stores via an effect on calcium-permeable channels, transporters, and exchangers on organellar membranes. Then, the change in cytosolic calcium concentration may trigger further distortion of the host cell system, which benefits virus survival and replication.The host cell plasma membrane is the first barrier against the invasion of viruses. Various CaThis review concentrates on host cell membranes’ calcium channels and pumps in viral infection. Blockers for these membrane proteins or preventing viruses from grabbing these host calcium-signaling components may lower the probability of virus stability, replication, and release, as well as infection-related host–cell apoptosis and reactive oxygen species production, neurotoxicity, and enterotoxin, making these membrane proteins potential targets for antiviral drugs.2+ is from two major sources: the internal Ca2+ store (mainly endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR)) and the extracellular medium. Calcium channels on cell plasma membrane mediate the entry of Ca2+ from the extracellular medium. These channels are activated by specific stimuli, such as voltage-gated calcium channels (VGCCs), which are stimulated by membrane depolarization, specific receptor-operated channels (ROC), which are stimulated by external agonists, or intracellular messengers and store-operated calcium channel (SOC), which are stimulated by the depletion of internal Ca2+ stores. The IP3 receptor (IP3R) and the ryanodine receptors (RyR) are the main players in mediating the release of Ca2+ from the internal stores. Inositol-1,4,5-triphosphate (IP3) activates IP3R, triggers Ca2+ release from stores, and further increases IP3R’s sensitivity to Ca2+. Calcium pumps (the plasma membrane Ca2+-ATPase (PMCA), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)) and the Na+/Ca2+ exchanger (NCX) are responsible for transporting Ca2+ from the cytosol to external medium or into cellular calcium stores  inhibited H1N1 and H3N2 IAV infection in multiple cell lines. IAV attaches to the target cells by the viral hemagglutinin protein (HA), which is a type I transmembrane protein embedded in the viral envelope, binding to sialic acids [V1.2, which contains two potential sialylated asparagine residues (N1436 and N1487) [V1.2 mutants in one or both these residues were replaced with glutamine , the interaction between HA and the Cav1.2 was attenuated compared with the wild-type fragment. They demonstrated that a VGCC blocker, diltiazem, significantly prolonged the survival of IAV-infected mice and allowed the recovery of the survivors. Therefore, CaV1.2 may serve as a host cell surface receptor that binds IAV and is critical for IAV entry  infection . Moreoveic acids . Fujiokad N1487) . When CaAV entry A. New woThe effect on VGCCs is not only restricted to virus entry. A high-throughput screening of an FDA-approved drug library for inhibitors of Japanese encephalitis virus (JEV) identified five hit drugs, three of which are VGCCs blockers  . Recombi2+ in various cell types, including neuronal, immune, and epithelial cells, via targeting the activity of VGCCs. The detailed information about the dysregulation of the L-type calcium channel by Tat can be found in the review [Further examples of virus-regulating VGCCs to service their replication can be found in HIV-1 and herpes simplex virus (HSV)-1. Two HIV-1 proteins, the membrane glycoprotein gp120 ,16 and te review . 2+ channels were reported as the targets of HSV-1 in sensory-like ND7-23 cells [2+ channels from the membrane, which depends on viral replication and protein synthesis. This downregulation of T-type Ca2+ channel expression may alter the ability of sensory neurons to transmit pain information. Thus, the lower expression of VGCCs may diminish the detection for viral infection by the host, which benefits virus survival and further infection.Taking together, the infections of viruses could increase the host’s intracellular calcium to facilitate viral entry and replication by manipulating host VGCCs. In addition, herpes simplex virus (HSV)-1 could downregulate the VGCCs on infected neuronal cells to escape detection by host cells. T-type Ca23 cells ,20. HSV-−/−, Tpcn2−/−) resisted EBOV infection [Ebola virus (EBOV) used to be considered a VGCC blocker-sensitive virus, and several research groups independently reported that compounds blocking L-type channels  inhibited EBOV infection in vivo ,22,23. Hnfection . It turnnfection ,28. The 2+ entry pathways in non-excitable cells. The protein Orai1 on the plasma membrane and STIM1  on ER are the molecular identities that are responsible for SOC channels activation. The depletion of ER Ca2+ stores promotes STIM1 proteins aggregation and interaction with Orai1 to open the channel, mediating Ca2+ entry [SOC channels are the major Ca2+ entry ,30. 2+ signal mediated by SOC channels (STIM1/Orai1).Most enveloped viruses are released extracellularly via exocytosis, also called budding, as an analogy of buds in plants ,32. The 2+ signals by activating the ER Ca2+ sensing protein STIM1 and the plasma membrane ORAI1 channel. The STIM1/Orai1-mediated Ca2+ signal is critical for EBOV and related viruses budding from host cells [2+ regulation [The research work done on four distinct hemorrhagic fever viruses (Ebolavirus (EBOV), Marburgvirus (MARV), Lassa Virus (LASV), and Junín Virus (JUNV)) demonstrated that EBOV, MARV VP40, and JUNV Z proteins trigger host cell Cast cells . The supgulation ,37. Ther2+ homeostasis possibly via promoting the interaction between STIM1 and ORAI1.Infections of other enveloped RNA viruses that buds in similar mechanisms may also be inhibited by STIM1 and ORAI1 inhibitors. Indeed, SOC channel antagonists significantly reduced DENV yield . When th2+ depletion in ER or prevent ER refilled with Ca2+ to maintain resting ER Ca2+ levels [2+ levels [It is possible that the viral proteins trigger the Ca+ levels ,41,42. A+ levels ,43. Thus+ levels . The mec2+ is the hallmark of rotavirus (RV) infection [Calcium influx through the SOC channel also contributes to the elevated cytosolic calcium concentration induced by rotavirus (RV) infection. It has been well established that the dramatically increased cellular Canfection ,46. Rotanfection ,48.2+ [The TRP channel is a non-selective cation channel predominately permeable for Ca2+ ,50. It i2+ signals in response to several stimuli, including hypotonic cell swelling, mechanical forces, moderating heat, and UVB radiation [2+ influx and drives the nuclear translocation of DEAD-box RNA helicase (DDX3X) [TRPV4 mediates intracellular Caadiation . When ce (DDX3X) . DDX3X i (DDX3X) ,54. More (DDX3X) . Overall (DDX3X) .The TRPV1, TRPA1, and TRPM8 channels are directly activated by chemical, thermal, and mechanical stimuli. They are potentially associated with respiratory virus-induced airway hypersensitivity . The inf3Rs are the two receptor-operated calcium (ROC) channels reported to be involved in virus–host interactions. NMDAr, which is activated by the endogenously synthesized excitant amino acid, glutamate, is widely expressed throughout the mammalian central nervous system and is particularly enriched in the cerebral cortex and hippocampus.NMDArs and IPThe Zika virus infection is a “neurodegenerative” disease. Costa and colleagues showed that blockage of the NMDAr channel activity with FDA-approved memantine or other antagonists prevents neuronal cell death and microgliosis induced by Zika in vitro and in vivo, without affecting the ability of Zika to replicate in the host ,59. It s2) to produce IP3. The activation of IP3R triggers Ca2+ release from stores and further increases IP3R’s sensitivity to Ca2+. Targeting IP3Rs is more universal among different virus types. In turn, this process generates a more profound effect on host cell physiology, including metabolic stress, neurotoxicity, and enterotoxicity. Modifying IP3 and directly interfering with IP3R are the main ways in which viruses affect calcium release through IP3R. For example, human cytomegalovirus (HCMV) UL37x1 protein interacts with the host P2Y2 purinergic receptor to increase intracellular Ca2+ levels via the PLC–IP3 pathway, and this activity is important to viral replication [3 [3R directly as an agonist. Glycoprotein E of HSV redistributes the density of IP3Rs within infected cells [3R through both ways. Enterotoxin NSP4 of RV and HCMV UL37x1-encoded protein increases the basal permeability of the ER as IP3 diffuses and binds to IP3Rs to stimulate Ca2+ release. These virus proteins deplete ER Ca2+ storage during early stages of viral infection to increase the replication ability of viruses. On the other hand, the depletion of ER calcium storage triggers the calcium influx through SOCs (STIM1/ORAI1).The engagement of receptors or agonist binding to the cell surface receptors activate phospholipase C (PLC), which hydrolyses phosphatidylinositol-4,5-biphosphate  and the Na+–Ca2+ exchanger are the main regulators of intracellular Ca2+ concentrations [2+ ATPase types (pumps) have been described in animal cells located in the membranes of the sarcoplasmic reticulum (SERCA pump), in those of the Golgi network , and in the plasma membrane (PMCA pump) [2+ concentration. For example, SERCA transports Ca2+ from the cytoplasm into the lumen of the ER. PMCA pumps function to remove Ca2+ from the cell. They serve as the main regulators of intracellular and extracellular Ca2+ concentrations. All three calcium pumps are reported to be involved in different viral lifecycles.The calcium pumps (Catrations . Three CCA pump) . At the 2+ pumped into Golgi by SPCA1 is the trigger to produce normal functional viral glycoproteins that are essential for virus spread. Therefore, SPCA1 may be a promising target for therapeutic intervention against a diverse set of viruses  infection ,70,71. F viruses B. HoweveCui et al.  screened2+ system by virus may suppress T-cell responsiveness, antiapoptotic, and other protentional functions. In this review, we summarized the calcium permeable channels and calcium pumps located on host cell membranes that may be potential therapeutic targets for viral infections (2+ binding proteins are targets for pharmacological intervention. Thus, explicating the entire picture of all aspects of the calcium–virus interplay at the molecular level will be a challenge in the future.The journey of viral infections is also a procedure during which a virus grabs the host intracellular calcium signaling system and homeostasis to benefit its own lifecycle. The disturbance of the host Cafections . HoweverThe prevention of and therapeutic efforts against virus infections are often challenged by the high mutation rates of viral proteins. However, those host calcium apparatus proteins required by viral replication and transmission are highly conserved, essentially immutable, and thus are potentially an Achilles’ heel of virus infection. As described above, almost all the calcium permeable channels or calcium pumps are possible attack targets of viruses and customized host intracellular calcium signaling via effects on these channel proteins. Targeting those host cellular proteins required by virus infection may be one treatment strategy with therapeutic potential, which may avoid or delay the development of resistance. The identified calcium-permeable proteins discussed above offer suitable target proteins for inhibiting virus infection. VGCCs and NMDA receptors are two of most well studied targets because of the availability of specific channel antagonists. Moreover, some of those compounds are FDA-approved drugs with clinical application. For example, verapamil, amlodipine, verapamil, and diltiazem, which are widely used to treat cardiovascular disease, have shown inhibition for various virus infection. Memantine, which is an FDA-approved Alzheimer’s drug, reduces neurological complications associated with Zika virus infection. Obviously, this direction depends on the identification of selective and potent small molecule modulators of calcium channels. Currently, aside from some blockers for VGCCs and the NMDA receptor, almost no other channel modulators can be used in clinics, although important progress has been made in recent years toward high quality and widely available channel modulators. Furthermore, modulators should be generated with properties that make them suitable for in vivo use. This review outlines promising progress made with the potentiation for a host cell calcium channels modulator to become an antiviral drug. We believe that modulators for host calcium channels are the pots of gold for antiviral drug development.


RYR2) that is responsible for highly penetrant, exertion-related sudden deaths/cardiac arrests in the Amish community without an overt phenotype to suggest RYR2-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). Homozygous RYR2 duplication (RYR2-DUP) induced pluripotent stem cell cardiomyocytes (iPSC-CMs) were generated from 2 unrelated patients. There was no difference in baseline Ca2+ handling measurements between WT-iPSC-CM and RYR2-DUP-iPSC-CM lines. However, compared with WT-iPSC-CMs, both patient lines demonstrated a dramatic reduction in caffeine-stimulated and isoproterenol-stimulated (ISO-stimulated) Ca2+ transient amplitude, suggesting RyR2 loss of function. There was a greater than 50% reduction in RYR2 transcript/RyR2 protein expression in both patient iPSC-CMs compared with WT. Delayed afterdepolarization was observed in the RYR2-DUP-iPSC-CMs but not in the WT-iPSC-CMs. Compared with WT-iPSC-CMs, there was significantly elevated arrhythmic activity in the RYR2-DUP-iPSC-CMs in response to ISO. Nadolol, propranolol, and flecainide reduced erratic activity by 8.5-fold, 6.8-fold, and 2.4-fold, respectively, from ISO challenge. Unlike the gain-of-function mechanism observed in RYR2-mediated CPVT, the homozygous multiexon duplication precipitated a dramatic reduction in RYR2 transcription and RyR2 protein translation, a loss of function in calcium handling, and a calcium-induced calcium release apparatus that is insensitive to catecholamines and caffeine.We identified a potentially novel homozygous duplication involving the promoter region and exons 1–4 of the gene encoding type 2 cardiac ryanodine receptor (  Molecular and functional characterization of the calcium release channel deficiency syndrome in patient-specific induced pluripotent stem cell-cardiomyocytes. RYR2 gene encodes for the protein known as the type 2 cardiac ryanodine receptor (RyR2)/calcium release channel (CRC), containing 4967 amino acids and 105 exons, that is a critical component of the normal calcium-induced calcium release (CICR) mechanism of excitation-contraction coupling in cardiomyocytes (CMs) (+) through the cardiac sodium channel (Nav1.5) leads to opening of voltage-dependent L-type calcium channels (LTCCs) during the plateau phase of the cardiac action potential. The entry of a small amount of Ca2+ into the cell triggers the massive release of Ca2+ from the sarcoplasmic reticulum (SR), the principal intracellular Ca2+ storage organelle of the cell, through RyR2s/CRCs. This cytosolic Ca2+ bathes the contractile filaments, binds to and activates protein troponin C (the Ca2+-sensing protein of the contractile apparatus), and induces myofilament contraction. Upon relaxation, the majority of the cytosolic Ca2+ is rapidly sequestered back into the SR by the SR Ca2+ ATPase (SERCA2a) or out into the extracellular space by the Na+/Ca2+ exchanger (The RyR2 gain-of-function (GOF) pathogenic variants account for 60% of autosomal dominant type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a potentially lethal, heritable arrhythmia syndrome that classically manifests as exercise-induced syncope, sudden cardiac arrest (SCA), or sudden unexplained death in the young (SUDY) in the setting of a structurally normal heart .RYR2’s 5′ UTR/promoter region, and exons 1–4 of RYR2 as a potentially novel genetic basis for recessively inherited, exercise-associated SCA/SUDY in 2, seemingly unrelated, large multigenerational Amish pedigrees  familial triangulation analysis, we identified the same large, homozygous, biallelic tandem duplication of 344,085 base pairs (bp) involving approximately 26,000 bp of intergenic sequence, n = 13, 5 male, 8 female; average age at death 11.7± 6.8 years) or survival of an SCA . Both sets of parents for patient 1 and patient 2 were heterozygous for the RYR2 duplication, were asymptomatic, and had normal cardiologic tests.Here, we characterize functionally patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) derived from 2 of the affected children, one from each pedigree . ClinicaRYR2 duplication as well as for 2 unrelated healthy controls who are negative for the RYR2 duplication. Two clones from each patient line  and 1 clone from each of the 2 controls  were used in this study. All 4 mutant iPSC clones tested demonstrated normal karyotypes  imaging using a confocal microscope (PBMCs were collected and used for iPSC generation for the 2 aforementioned unrelated patients who survived an exertion-associated SCA and are homozygous for the ryotypes . Sanger ectively . Undiffecroscope . A convecroscope . The oveRYR2 transcript was reduced significantly to just 31.5% ± 4.5% (P < 0.004) of control levels in the RYR2 duplication iPSC-CMs from both affected individuals  who died suddenly during exertion . Comparel levels . Westernl levels . Using ql levels , while tl levels .2+ imaging. The [Ca2+]i transient amplitude normalized by (F – F0)/F0, the 50% [Ca2+]i transient decay time, and the [Ca2+]i transient upstroke time to peak in the iPSC-CMs from both patient RYR2 duplication lines were indistinguishable from WT control iPSC-CMs under baseline conditions  and WT control iPSC-CMs (WT1 and WT2) were tested with Fluo-4 based Canditions .2+ recording to discern any intracellular calcium handling abnormalities during β-adrenergic stimulation. As expected, 100 nM ISO elicited shortening of the [Ca2+]i transient upstroke peak time and [Ca2+]i transient decay time in WT control iPSC-CMs /F0 was observed in the WT control iPSC-CMs , the patient RYR2 duplication iPSC-CMs  did not elicit any normal ISO-mediated amplitude change   was used in Fluo-4 CaiPSC-CMs  and bothiPSC-CMs . While t to ISO) .2+ storage/release capabilities of the mutant and control iPSC-CMs using caffeine-mediated (10 mM) RyR2 activation. Both control iPSC-CMs (WT1 and WT2) showed the expected abrupt and dramatic increase in intracellular Ca2+ amplitude after caffeine treatment  were not observed in any control iPSC-CMs . HoweverP = 0.004) and 17% of RYR2 Dup 2-c1 (P = 0.005) wells showing erratic activity in response to 100 nM ISO challenge . One week after plating iPSC-CMs in a CardioECR multielectrode plate, a recording of field potential with a minimum of 0.1 mV amplitude could be obtained . There whallenge . We alsoiPSC-CMs . Both RYting CMs .+ channel blocker also used for the treatment of CPVT1, was tested in our current setting. Flecainide appeared to be slightly less potent than the beta blockers but had a significant rescue effect in arrhythmia, with a 2.4-fold reduction in erratic activity during ISO challenge  that also leads to an overall GOF of RyR2 have been reported to cause a wide spectrum of clinical phenotypes, including autosomal dominant CPVT1, sinoatrial node dysfunction, bradycardia, atrial fibrillation, atrioventricular block, dilated cardiomyopathy, left ventricular noncompaction, and SCA  being symptomatic and 18/23 (78%) dying suddenly  or polymorphic VT , 4/30 (13%) probands were classified as having “catecholaminergic idiopathic ventricular fibrillation (cIVF),” defined as ventricular fibrillation elicited by physical or emotional stress in the absence of an identifiable precipitating factor and in the absence of VT documented at Holter and/or exercise stress testing , a 7-year-old girl presenting with syncope, had the p.A4860G-RyR2 variant that was demonstrated most recently to confer a loss of function (LOF) to the RyR2/CRC and lead to the formation of early afterdepolarizations (EADs) . In fact2+ responsiveness to ISO, and dramatically reduced Ca2+ responsiveness to caffeine (RyR2 agonist).Indeed, rather than a GOF in calcium release through hyperactive or hyperreactive  RyR2 calcium release channels as typically observed in CPVT1, our patient-specific homozygous RYR2 duplication iPSC-CMs displayed a cellular phenotype of CRC deficiency, as evident by severely reduced RyR2 protein levels, essentially no CaRYR2 transcript and RyR2 protein levels. In fact, we observed a 70% to 80% reduction in RYR2 mRNA transcript and RyR2 protein expression levels in both homozygous RYR2 duplication iPSC-CMs compared with control iPSC-CMs. The substantial reduction in RyR2 protein expression was confirmed in an autopsy sample of heart tissue from a 12-year-old homozygous RYR2 duplication–positive female relative who died suddenly while playing.We have demonstrated that the large homozygous biallelic tandem duplication identified in our pedigrees leads to a significant reduction in KCNQ1, KCNH2, SCN5A, and RYR2, uncommonly serve as the pathogenic basis of dominantly inherited arrhythmia syndromes such as long QT syndrome, Brugada syndrome, and CPVT , a T-box transcription factor–dependent long noncoding RNA located 25,000 bp upstream of RYR2, was required for RYR2 gene expression (RYR2 gene and has a similar role in humans. Our patient-specific human iPSC-CM (hiPSC-CM) lines may allow us to further investigate the regulatory role of this duplicated region in the control of RYR2 gene expression.Interestingly, Yang and colleagues reported in mice that pression . It is n2+ sparks or ectopic beating in control iPSC-CMs and RYR2 duplication iPSC-CMs (data not shown), we did observe a substantially reduced Ca2+ responsiveness to β-adrenergic stimulation by ISO. In control iPSC-CMs, we saw the expected increase in Ca2+ transient amplitude. The ISO-mediated Ca2+ peak time and decay time response was indistinguishable between control and patient iPSC-CMs. However, the amplitude response was reduced dramatically (no response to ISO) in patient iPSC-CMs in both independent patient lines compared with control cells. Yet, the peak upstroke time and Ca2+ decay time, which translated as activation of RyR2 by PKA and SERCA2a, was not different between control and patient iPSC-CMs. This indicates that the RYR2 channels are functional in the homozygous RYR2 duplication patient lines but consistent with lower RyR2 protein levels than in control lines.While we did not notice a difference in ISO-induced Ca2+ responsiveness to caffeine similar to what has been observed in our RYR2 duplication iPSC-CM lines. However, Bround and colleagues, using a heart-specific inducible RYR2-haploinsufficient (cRyr2Δ50) mouse model with only a single null allele, saw no difference in caffeine-releasable Ca2+ between the cRyr2Δ50 and control CMs  in RyR2 protein. In fact, both the patient iPSC-CMs and the heart tissue from one of the sudden death victims revealed a 70%–80% reduction in RyR2 protein.Our control iPSC-CMs displayed the expected abrupt and dramatic increase in intracellular calcium following caffeine treatment. However, neither RYR2 duplication hiPSC-CM line had any significant change in calcium transient amplitude following caffeine. Although RyR2-null mice are embryonically lethal, rodent RyR2-knockout embryonic stem cell–based  and RyR2trol CMs . Importa2+ responsiveness to caffeine observed in our homozygous RYR2 duplication iPSC-CMs appears different from previous HEK293 cell–based analysis of the aforementioned LOF-associated missense variants, where both p.A4860G and p.S4938F exhibited a normal caffeine response  or length of suppression of siRNA during experimental measurement (72 hours or 1 week after siRNA treatment). This may suggest a certain threshold of RYR2 transcript/RyR2 protein loss that is required before observing a pathogenic phenotype. The behavior of erratic activity observed in the RYR2 duplication iPSC-CMs seemed to mimic that of DAD activity, which was elicited after full FP generation. Currently, triggered DADs are considered the most accepted cellular mechanism to explain arrhythmias in CPVT . While not consistently observed within the 2 Amish pedigrees, some individuals homozygous for the RYR2 duplication have been noted to display intermittently prolonged QT intervals  . For patRYR2 duplication mutation identified in 2 large Amish pedigrees associated with exertion-related SUDY/SCA. Unlike the typical GOF mechanism observed in RYR2-mediated CPVT, the homozygous multiexon duplication precipitates a “calcium release channel deficiency syndrome” secondary to a profound attenuation in RYR2 transcription and RyR2 translation, which renders the CICR apparatus nearly insensitive to both catecholamine stimulation and caffeine.Here, we characterized what we believe is the first iPSC-CM model of a homozygous multiexon RYR2 duplication. Human iPSCs were generated from PBMCs derived from 2 unrelated patients (15-year-old girl and 10-year-old boy) who either survived an exertion-associated SCA and/or have an extensive family history of multiple SUDYs. iPSCs were also generated for 2 unrelated healthy female controls who were negative for the RYR2 siRNAs were purchased from OriGene Technologies, Inc.  and 1 scramble RNA . The siRNAs were activated by adding RNase-free duplex buffer based on the manufacturer’s recommendation . Using Lipofectamine RNAiMAX , 20 μM final siRNAs were transfected into control WT1 iPSC-CMs and incubated for 72 hours based on the manufacturer’s recommendation.Two human RYR2 and an endogenous control (GAPDH) gene were assessed using the 7900 RT-qPCR system . Relative gene expression analysis was performed using a modified ΔΔCt method employing the Pfaffl formula, which accounts for PCR amplification efficiencies between primer sets. Statistical analysis was performed using GraphPad Prism (GraphPad Software).Following physical enrichment of iPSC-CMs from beating clusters, total RNA was isolated from multiple biological replicates from all 3 homozygous RYR2 duplication mutant human iPSC-CM clones and control iPSC-CMs. Gene expression levels for Western blot analysis was performed on protein lysates isolated from WT and mutant iPSC-CMs following the detailed methodology outlined in the To demonstrate that the CMs expressed the typical cardiac markers, IF staining for cTnT, cardiac RyR2, and DAPI was performed using the detailed methods outlined in the supplemental materials.Frozen sections of human heart were processed by the Mayo Histology Core lab. Each frozen section was fixed with a cold (–20°C) acetone  and methanol  (50:50) mixture for 5 minutes at –20°C. The sections were washed immediately at room temperature with PBS to rehydrate tissue. Blocking and primary/secondary antibody staining followed as described above and in the supplemental materials. Imaged data were analyzed by ZEN lite software (ZEISS) to quantify the average intensity that was collected and were analyzed by the statistics software GraphPad Prism (GraphPad Software).2+ load and RyR2-mediated leakage were assayed using Fluo-4 fluorescence. Patient and unrelated control iPSC-CMs were dissociated from 24-well plates and then plated and cultured on Geltrex-coated , 35 mm, glass-bottom dishes  at 37°C, 5% CO2, for 3–5 days. CMs were incubated for 30 minutes in DMEM/2% FBS media containing 2 μM of Fluo-4 AM  and 0.02% F-127 . The cells were washed once with fresh DMEM/2% FBS media. During imaging, the dishes were kept in a heated 37°C stage-top environment chamber supplied with 5% CO2. Imaging of Ca2+ transients was taken under ×40 objective using Nikon Eclipse Ti light microscope. Ca2+ transients from single beating iPSC-CMs, paced at 0.25 Hz, were recorded at a speed of 20 ms per frame for 20 seconds at 15% LED power. After finishing baseline recording, appropriate amounts of ISO  and caffeine  were added into the recording dish dropwise. The raw data were exported to Excel software (Microsoft) and then analyzed with an in-lab–developed Excel-based program.CM SR Ca2 incubator at 37°C before use.CM aggregate cultures were maintained in DMEM. At differentiation days of 30–60, the iPSC-CMs were subjected to enzymatic dissociation to obtain single-cell suspensions of CMs. These cells were added to 0.1% gelatin-coated glass coverslips maintained in DMEM and stored in a 5% CO2, 1 MgCl2, and 10 HEPES, pH adjusted to 7.4 with NaOH. The pipette solution contained (mmol/L): 110 KCl, 31 KOH, 10 EDTA, 5.17 CaCl2, 1.42 MgCl2, 4 MgATP, and 10 HEPES, pH adjusted to 7.2 with KOH. Microelectrodes were pulled on a P-97 puller (Sutter Instruments) and fire polished to a final resistance of 2–3 MΩ. Series resistance was compensated by 80%–85%. Data were analyzed using Clampfit (Axon Instruments) and Excel and plotted with Origin 9.1 (OriginLab Corporation).Action potentials from control and RYR2 Dup 1 and RYR2 Dup 2 mutant iPSC-CMs before and after ISO were recorded at room temperature (22°C–24°C) using current clamp mode at a constant rate of 1 Hz through 5 ms depolarizing current injections of 150–500 pA or gap-free mode with the use of an Axopatch 200B amplifier, Digidata 1440A, and pClamp version 10.2 software (Axon Instruments). The extracellular (bath) solution contained (mmol/L) 140 NaCl, 4 KCl, 2 CaCl2O, and was diluted into aqueous bath solutions to achieve final concentrations of 100 nM or 1 μM.ISO hydrochloride was purchased from MilliporeSigma, was prepared as 100 μM stock solution in H2. FPs were measured 2 times for 20 seconds (block duration) every 10 minutes as a routine baseline measurement.Arrhythmic activity was assessed using an xCELLigence RTCA CardioECR instrument (ACEA Biosciences) at baseline (DMEM/2% FBS media) and during ISO challenge (100 nM). Patient and unrelated control iPSC-CMs were dissociated from 24-well plates and then plated and cultured on fibronectin-coated , 48-well, electronic microtiter plates  at 37°C, 5% COVarious pharmacotherapies were used to test their efficacy on the treatment of arrhythmic events occurring in the mutant iPSC-CMs as a proof-of-principle, treatment-in-a-dish pilot study. Propranolol (1 μM), nadolol (10 μM), or flecainide (6 μM) was administered in combination with ISO (100 nM) immediately before recording. For the drug treatment test, a 1-hour setting was used with a 5-minute interval and 20-second block duration. The percentages of wells presenting arrhythmic activity were recorded per treatment. ISO, propranolol, nadolol, and flecainide were purchased through MilliporeSigma.t test was performed to determine statistical significance between 2 groups, and a 1-way ANOVA was performed for comparisons among 3 groups. For multiple post hoc ANOVA analyses, both Tukey’s and Bonferroni’s tests were used. P < 0.05 was considered significant.Data points are shown as the mean value, and bars represent the standard deviation unless specially mentioned. A Student’s 2-tailed Human iPSCs were generated from 2 unrelated patients (15-year-old girl and 10-year-old boy) following receipt of written informed consent for this Mayo Clinic IRB–approved study (09-006465). The heart tissue sample was used following written informed consent from the deceased family member’s parent for this Mayo Clinic IRB–approved study (1216-97).DJT contributed to conceptualization of the project, designing of research studies and experiments, data analysis, and writing of the manuscript. CSJK contributed to conceptualization of the project; performed most of the experiments; and contributed to experimental design, data acquirement and analysis, and writing of the manuscript. SKH, DY, and BJO contributed to data acquirement and analysis and writing of the manuscript. HMB, KKF, CMHT, DLA, LAON, IL, and JT contributed to patient sample recruitment, clinical information gathering, and critical review of the manuscript. MJA contributed to conceptualization of the project, designing of research studies and experiments, and writing of the manuscript. All authors discussed the results and commented on the manuscript.


However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.Severe immunodeficient mice are widely used to examine human and animal cells behaviour  In particular, immunodeficient mice are most often used because of their high reproductive ability and ease of handling. Nude mice with a Foxn1 mutation have been widely used in studies on cancer as athymic mice2. Thereafter, SCID mice with Prkdc mutations were discovered3, which succeeded in differentiating human haematopoietic stem cells in the mouse body upon transplantation of human foetal liver and thymus tissues under the renal capsule; these mice are referred to as SCID-hu and are widely used in studies on HIV4. Il2rg-mutant mice have recently been developed through gene targeting technology5; this gene is essential for signal transduction among interleukins such as IL-2, IL-4, IL-7, IL-9, and IL-15.Immunodeficient animals are important for Il2rg is located on the X chromosome, and a mutation of the γ chain in humans causes X-SCID6. Examples of X-SCID-animals include rats7, marmosets8, pigs9, and dogs with the gene mutation10. Furthermore, functional defects such as T, B, and/or NK loss lead to severe immunodeficiency.in vivo evaluation. In contrast, large animals such as pigs and monkeys can have a longer lifespan and larger organ size; however, they require advanced handling expertise and a large-scale facility, resulting in a higher cost. Moreover, large animals have a long period of sexual maturity and gestation period.Although numerous mouse models of immunodeficiency have been reported and handling of animals is relatively easy, they are small in size and short-lived, thus complicating surgical procedures in certain regions including the eye and blood vessels and resulting in inadequate long-term 11. Hence, rabbits are regularly employed in studies of infectious disease12, liver cancer13, adult T cell leukaemia-lymphoma14, joint or bone disease16, dentistry17, eye disease , cardiovascular disease21, atherosclerosis24, and lipid metabolism25 and for the production of antibodies26. The detailed rabbit genome has recently been published in the Broad Institute server (https://www.broadinstitute.org/rabbit/rabbit-genome-project) and RGB-Net: Collaborative European Network on Rabbit Genome Biology (http://www.biocomp.unibo.it/rabbit/index.php). Numerous studies have reported rabbits as models of human disease through genetic modification29.Rabbits can be operated on relatively easily owing to their size. Furthermore, rabbits are the second most widely used animal after experimental rodent models owing to their long life, ease of handling, and the ability to be reared in a small space. Further, they have a short period of sexual maturity and gestation and are more genetically related to humans than other rodents31 have been developed, no study has functionally analysed the lineage stability of Il2rg gene-deficient rabbits. This study reports the development of Il2rg-KO rabbits and maintenance of a stable strain, using the CRISPR/Cas9 system.Although several rabbit models of immunodeficiencyCas9 and gRNA concentrations were divided into two conditions: low and high concentrations Table . At low Il2rg locus despite having only one X chromosome, suggesting that mosaicism was induced upon CRISPR/Cas9 treatment, a situation frequently observed in the DNA of transgenic founders. Two of the three female offspring harboured biallelic mutations. We maintained the strain with the 265-bp deletion as it was easy to identify gene disruption and numerous amino acid deletions were expected.Three of the five male offspring harboured mosaicism biallelic mutations at the Il2rg−/Y) rabbits display the severe immunodeficiency phenotype (X-SCID); however, heterozygous female (Il2rg−/+) rabbits display a normal phenotype32. Therefore, hemizygous male  and WT female rabbits were mated to produce heterozygous female (Il2rg−/+) offspring with a 265-bp deletion in the F1 generation  profiles, including the white blood cell (WBC) count, were lower than those in WT rabbits Table , and difFlow cytometry analysis of cell populations isolated from peripheral blood mononuclear cells revealed a marked reduction in the number of lymphocytes in X-SCID rabbits Fig. . The numRT-PCR analysis revealed a reduction in mRNA size in the spleen of X-SCID rabbits in comparison with WT rabbits Fig. .Gross and microscopic analyses revealed that X-SCID rabbits underwent abnormal lymphoid development. In the thymus, it was difficult to isolate the exact thymus tissue from X-SCID rabbits for measuring the weights because of the remarkable thymus hypoplasia. Instead, we examined thymus tissue by HE-staining, indicating that thymus from X-SCID rabbits contained almost no lymphocytes Fig. . AlthougFor skin transplantation, Dutch rabbits  were used as donors and X-SCID rabbits  and WT rabbits  were used as recipients Fig. . No markHistological examination via haematoxylin and eosin staining in X-SCID rabbits revealed marked fibroblast proliferation at the interface between the transplanted and original skin, and no inflammatory cell infiltration, including lymphocytes, was observed. However, in WT rabbits, numerous lymphocytes were observed at the interface Fig. . During Il2rg-KO rabbits can be generated with a high genome editing efficiency of 46.7%. The efficiency was relatively high compared to that of Il2rg-KO model mice5 , and rats7, , rabbits31, , pigs9, , and marmosets8, i.e., . In a previously generated rabbit model of immunodeficiency30, the genome-editing efficiency was 100%; however, all founder rabbits died in 45 days. Our X-SCID rabbit model and the previously generated rabbit model of immunodeficiency31 were subjected to the same Il2rg gene KO; however, the target sites were different, thus also being associated with genome editing efficiency and lineage stability. Non-rodent experimental animals including rabbits are often used for long-term follow-up evaluation studies such as transplantation experiments. Rabbits yield offspring over a short period, with 4–5 months of sexual maturity and a gestational age of approximately 30 days in comparison with other large animals 33. The ability to obtain highly efficient progeny (F1/F2) that can rectify mosaic mutations and epigenetic defects in the 6th month in the shortest period leads to shortening of the study period, which is advantageous for research and development34.This study reports the development of established X-SCID rabbits via the CRISPR/Cas9 system and their stable maintenance, demonstrating that Il2rg−/Y (X-SCID) rabbits herein presented marked hypoplasia of the thymus, a decreased lymphocyte count in the white pulp area in the spleen, loss of peripheral lymph nodes, and a significant reduction in the lymphocyte count in peripheral blood, similar to other X-SCID animals. These observations indicate that in rabbits as well, the Il2rg plays contributes to the development and differentiation of lymphocytes and its functional deficiency induces immunodeficiency symptoms. X-SCID rabbits displayed a significant reduction in the number of CD3+ T cells among peripheral blood mononuclear cells during sexual maturation. In X-SCID mice and marmosets, T cells increase in infancy 35. In human X-SCID, CD3+ T cells are almost completely obliterated6; hence, rabbit phenotypes can be considered similar to those of humans. Furthermore, the number of B cells remains constant in WT pigs and human X-SCID9; however, the number of B cells is significantly reduced in mice and rats7. Herein, the number of IgM+ B cells also significantly reduced in the rabbits, displaying the same tendency as that of mice and rats. NK cells were not detected as no specific antibody to rabbit NK cells was identified, as previously reported31. T/B-cell fractions differ among animal species with age. Herein, T and B cells were significantly reduced during sexual maturation of rabbits; however, in X-SCID rabbits, further detailed subset analysis of T and B cells in each immune organ and further analyses are required to elucidate the development and differentiation of T and B cells.The 37, and skin transplantation is a non-invasive method. In our skin transplantation experiments, we used Dutch rabbits as donors and Japanese White rabbits (X-SCID rabbits) as recipients. Dutch rabbits have black hair, while X-SCID rabbits have white hair; hence, it is easy to ascertain graft acceptance. The Dutch rabbits belong to the Dutch germline Oryctolagus cuniculus; while the Japanese White species belong to Japanese germline O. cuniculus. The genomes of Dutch and Japanese White rabbits belong to different groups38. Although the innate immune system was functional in the X-SCID rabbits, we were able to confirm the engraftment of allogeneic skin in these rabbits. T and B cells are largely involved in the rejection of transplanted immunity and it has been shown that both the number and function of T and B cells are decreased in X-SCID rabbits. The degree of rejection varies depending on the type of organ (tissue), and among them, skin tissue is susceptible to rejection39. Therefore, survival can potentially be achieved in the transplantation of organs (tissues) other than the skin. Furthermore, in X-SCID pigs9, lymphoid cell reconstruction was confirmed via allogeneic bone marrow transplantation, suggesting that X-SCID rabbits with the same phenotype can be expected to be used as a model for immune system reconstruction through bone marrow transplantation.Furthermore, we performed allogeneic transplantation and confirmed skin engraftment among individuals with genetically different characteristics. Skin transplantation is a well-established method often used in studies on the immune response and rejection9, probably owing to antibiotic administration and housing conditions. Antibiotics were used from 6 weeks after weaning, and the previous studies on X-SCID rats41 have reported that the infection was suppressed through antibiotic administration. Furthermore, in a previous study, rabbits were maintained under conventional housing conditions30, in contrast, our rabbits were maintained in a clean booth until they were approximately 9–15 weeks old. Furthermore, inbreeding degeneration is rapid among rabbits40; however, the issue was resolved through mating between a heterozygous individual with the same phenotype as that of WT and a WT fertile individuals raised in another colony. From the F2 generation, pneumonia symptoms and diarrhoea, which are considered to be caused by Pneumocystis oryctollagi, were observed. This condition has also been reported in X-SCID rats and other immunodeficient rabbits42. A previous study reported42 that before weaning, X-SCID rabbits were resistant to Pneumocystis owing to immunity acquired from the mother; however, severe infection was observed sometimes after weaning. These results suggest that the lung abnormality did not result from gene knockout itself but rather from Pneumocystis pneumonia due to a suppressed immune response after weaning. Furthermore, WT rabbits harbouring an antibiotic specific for Pneumocystis pneumonia have reduced P. oryctollagi and the individuals are used as parent WT rabbits, and the offspring are obtained through caesarean sections and reared under aseptic conditions, resulting in the suppression of Pneumocystis infections in the same manner as X-SCID rats45. Thus, we speculate that X-SCID rabbits survive longer and efficient strain maintenance is possible.We also investigated whether X-SCID rabbits can stably maintain their lineages. Consequently, we obtained a stable line of rabbits up to the F4 generation. The average lifespan of the F2 generation X-SCID rabbits was approximately 200 days, and the X-SCID pigs survive for a longer period than the average period of approximately 70 days46. X-SCID rabbits with the same phenotype can be expected to be used as humanised animal models transplanted with human-type organs (tissues) or pluripotent stem cells. Furthermore, the number of transplantable cells is limited because Il2rg-KO mice and X-SCID rats have a small body size. X-SCID rabbits can be transplanted with a similar or equal number of cells to that of humans. Safety tests including those related to regenerative medicine and cell therapy are potentially applicable because they are considered advantageous for optimising the dosage of transplanted cells and for confirming the characteristics of grafted cells at the transplant site. Accordingly, rabbits are potentially applicable as a link between mice and rats and other animal species including pigs and monkeys, and as an excellent tool for studies on organ and cell transplantation, cancer, and haematopoietic system bridging to humans.We thus generated highly immunodeficient animals. X-SCID rats were confirmed with regards to human ovarian cancer cells and survival of human embryonic cells or human induced pluripotent stem cell (iPSC)-derived DA neuronsJapanese White (JW) rabbits were obtained from KITAYAMA LABES Japan  and housed under conditions of 30–70% humidity and a 12:12-h light/dark cycle. They were fed with pellets which had been sterilised via gamma irradiation (50 kGy/box) and supplied with water treated using a reverse osmosis system . All X-SCID rabbits were subcutaneously injected twice per day with 10 mg/kg enrofloxacin from six weeks of age.All animal care and experimental protocols conformed to the Guidelines for Animal Experiments of Osaka University and were approved by the Animal Experimental Committee of Osaka University. All of the experiments using recombinant DNA were approved by the Recombinant DNA Committees of Osaka University and were performed in accordance with our institutional guidelines.in vitro using a mMESSAGE mMACHINE T7 Ultra Kit  and was purified using a MEGAClear kit (Thermo Fisher Scientific) and resuspended in RNase-free water, in accordance with the manufacturer’s instructions.A Cas9 expression plasmid (RDB13130) was used herein. To generate a Cas9 mRNA expression vector, plasmids were linearized with Nhe1 , and extracted with NucleoSpin Gel and PCR Clean-up kits  in accordance with the manufacturer’s instructions. After purification of the linearized DNA, Cas9 mRNA was transcribed in vitro using a MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions from synthetic double-stranded DNA containing a T7 promoter, 20-bp target sequences, and gRNA tail sequences . The gRNA DNA template was synthesised in vitro using CUGAR 7 in vitro Transcription Kit  in accordance with the manufacturer’s instructions and transcribed. mRNA was purified using a MEGAClear kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions and resuspended in RNase-free water48.gRNA was designed using CRISPR DESIGN software (http:crispr.mit.edu/). gRNAs were transcribed Sexually matured JW female rabbits , as donors, were super-ovulated via subcutaneous injection of follicle-stimulating hormone  six times per day. Seventy-two hours after FSH injection, the donor females were artificially inseminated, and a single dose of 75 IU human chorionic gonadotropin  was immediately intramuscularly administered to induce ovulation. Simultaneously, 75 IU of hCG was injected to sexually mature recipient female rabbits to induce pseudopregnancy. Nineteen to 21 h after hCG injection, fertilised eggs were harvested from donor females and pronuclear-stage embryos were selected and cultured in a modified medium  supplemented with 5% FBS  before the microinjections. Using a micromanipulator , 200 ng/uL Cas9 mRNA, 100 ng/ul gRNA, 15 ng/ul pExo1 or 100 ng/ul Cas9 mRNA, 50 ng/ul gRNA, 15 ng/ul pExo1 were microinjected into the male pronuclei of embryos. These embryos were cultured in a modified medium  supplemented with 20% FBS  after the microinjections. Surviving embryos were sorted and twenty to thirty embryos were transplanted into the oviduct of recipient rabbits. A part of the tail of few-day old pups tail was sampled and genome DNA was extracted using the KAPA Express Extract Kit . PCR was performed using the following conditions: 94 °C for 1 min, 35 cycles of 98 °C for 10 s, 61 °C for 15 s, and 68 °C for 30 s, and final extension at 72 °C for 1 min Table . The PCRBody weights of rabbits (F2 offspring X-SCID [n = 5] and WT [n = 5]) were assessed once a week. Rabbits were monitored throughout the observation period and mortalities were recorded daily (F2 offspring X-SCID [n = 5] and WT [n = 5]). Blood parameters were analysed in 20-week-old, X-SCID (n = 4) and control WT (n = 4) F2 offspring. Approximately 600 μL peripheral blood was sampled from the ear vein. Blood parameters including the blood cell count and a differential WBC count were measured using ADVIA2120 .Il2rg-/Y (n = 4–5) and control WT (n = 5–6) F2 offspring. To enumerate T and B lymphocytes in whole blood, approximately 2 mL of peripheral blood was sampled from the ear vein in a heparin-coated syringe. Lymphocytes from blood were separated using a Ficoll-Paque PREMIUM  gradient via centrifugation for 40 min at 400 × g. Cells were filtered with a 40-μm cell strainer  and washed with PBS to eliminate debris. The remaining pellets were resuspended in 1 ml of PBS and enumerated with a haemocytometer. Anti-human CD3 , anti-rabbit IgM  were used as primary antibodies. Mouse IgG1 antibodies  were used as isotype-matched controls. For intracellular protein staining, a BD Cytofix/Cytoperm (BD Biosciences) kit was used in accordance with the manufacturer’s instructions.Flow cytometric analyses were performed for 17–25-week-old, 5 cells per sample were incubated with the primary antibodies in PBS for 30 min on ice and washed twice via centrifugation at 400 × g for 5 min. Thereafter, cells were incubated with Alexa Fluor 647-conjugated secondary antibodies  under the same conditions. After washing, the cells were resuspended with 500 μL PBS before FACS analysis. Thereafter, 10,000 live cells were used for flow cytometry analysis using FACSVerse (BD Biosciences). The data were analysed using the BD FACSDiva Software (BD Biosciences).Approximately 2.5–5.0 × 10Gapdh was used as an internal control refer to previous study49.Total RNA was extracted from the spleen of 36-week-old F2 offspring using QIAzol reagent . Reverse transcription was performed using the SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol, and cDNA was used as a template for PCR. The cycling conditions were as follows: 98 °C for 10 s, followed by 40 cycles at 60 °C for 15 s and 68 °C for 30 s Table . PCR proThe rabbits were anaesthetised via isoflurane inhalation 4–5% and maintained at 2–3%. The dorsae of the rabbits were shaved, and the surgical area was disinfected with Povidone-Iodine and washed with 70% ethanol using sterile cotton, and finally dried with sterile gauze. The surgical procedure was carried out under aseptic conditions.2 area was marked and harvested using scissors and a scalpel. Immediately thereafter, the grafts stored in HBSS (Thermo Fisher Scientific) at 4 °C until grafting. The recipient rabbits were inflicted with a full-thickness excision skin wound of 1.5 × 1.5 cm2 on the dorsa, using a scalpel and scissors. The skin graft was grafted at the dorsal wound in recipient rabbits, using 5–0 PROLENE  sutures, with one stitch at each of four corners and 3–4 stitches on each side. The skin graft sites were covered with a transparent dressing , and sterile gauze, elastic bandage, and surgical tape were applied to seal the wound. Upon regular inspection of the wounds, the bandages were changed50.The skin graft donor site was located on the dorsa and a 1.5 × 1.5-cmStitches and bandages were removed on day 14, and the skin grafts were inspected on days 0, 1, 3, 5, 7, 10, 12, 14, and 21 after surgery. Two X-SCID rabbits were inspected up to day 56. Graft rejection was detected through the macroscopic appearance of graft necrosis, while graft necrosis was evaluated using a scale based on colour changes and scarring. At 21–56 days after transplantation, the animals were euthanised with pentobarbital. Graft skin tissues were fixed in 10% formalin solution, embedded in paraffin, and sectioned. The sections were stained with haematoxylin and eosin.The grafted skin tissues were embedded in Tissue-Tek® Optimal Cutting Temperature compound  and sectioned at 10-μm thickness. The sections were washed thrice with Tris-buffered saline  for 10 min and blocked for 1 h in TBS (TaKaRa Bio) containing 5% donkey serum  and 0.3% Triton X-100 . The sections were then probed overnight at 4 °C with anti-CD3  and anti-CD45  antibodies in TBS containing 1% donkey serum and 0.3% Triton X-100. Subsequently, samples were again washed thrice with TBS for 5 min and were labelled for 1 hour at room temperature (20 °C–28 °C) with secondary antibodies conjugated with Alexa Flour 488 (Thermo Fisher Scientific). The sections were counterstained with Hoechst 33342 (Thermo Fisher Scientific) to visualise the nuclei and imaged using a fluorescence microscope .Haematoxylin and eosin staining, tissues were fixed with 10% formaldehyde neutral buffered solution and embedded in paraffin and then 3–5-μm-thick sections were cut and stained with haematoxylin and eosin after deparaffinisation and hydration. The sections were imaged using a microscope equipped with a NanoZoomer-XR C12000 system  and an Axio Observer.D1 system. Grocott staining was performed by KAC Co., LTD. .All data are expressed as mean ± S.D. values. Statistical analyses were performed using the Welch’s t test. p values of <0.05 were considered to indicate statistical significance. All of statistical analyses were performed using the GraphPad Prism8 software .Supplementary dataset 1.


Extracellular vesicle (EV) trafficking provides for a constitutive mode of cell-cell communication within tissues and between organ systems. Different EV subtypes have been identified that transfer regulatory molecules between cells, influencing gene expression, and altering cellular phenotypes. Evidence from a range of studies suggests that EV trafficking enhances cell survival and resistance to chemotherapy in solid tumors. In acute myeloid leukemia (AML), EVs contribute to the dynamic crosstalk between AML cells, hematopoietic elements and stromal cells and promote adaptation of compartmental bone marrow (BM) function through transport of protein, RNA, and DNA. Careful analysis of leukemia cell EV content and phenotypic outcomes provide evidence that vesicles are implicated in transferring several known key mediators of chemoresistance, including miR-155, IL-8, and BMP-2. Here, we review the current understanding of how EVs exert their influence in the AML niche, and identify research opportunities to improve outcomes for relapsed or refractory AML patients. Acute myeloid leukemia (AML) is a genetically heterogenous disease that arises from abnormal proliferation of hematopoietic stem cells (HSCs) . AlthougExtracellular vesicles (EVs) are membrane-bound particles secreted from cells, carrying a variety of nucleic acid and protein cargo active in cell-cell communication . As a coAML is a genetically heterogeneous disease characterized by the successive acquisition of mutations in HSCs that cause unchecked proliferation and a coincident differentiation arrest . At diagDevelopment of drug resistance during chemotherapy is a primary challenge to sustaining AML remission in patients. This can occur as a direct consequence of mutations, or indirectly through signaling pathways or enzyme activities that lead to cancer cell protection . ConceptNon-genetic chemoresistance mechanisms such as p-glycoprotein (P-gp) overexpression, an ATPase efflux pump that export drugs or their active metabolites, are also correlated with poor disease outcomes . These dEvidence has also shown that cellular microRNAs are highly involved in the development of drug resistance in AML . For exaThe bone marrow compartment comprises a range of cell types that form HSC supportive microenvironments: endosteal  and vascular  niches . The endEmerging evidence also suggests that AML blasts localize and integrate with the BM vascular niche. A study by Cogle et al. showed that some AML cells have the capacity to fuse with vascular endothelium (vascular-AML), acquiring several endothelial characteristics before assuming a state of quiescence . MoreoveIgf1, Cxcl12, Kitl, and Il-7), and genes involved in normal bone development (Ocn and Col1A1). In addition to playing a key role in transforming the BM niche, exosomes may in part contribute toward the protection of leukemia blasts during chemotherapy treatment  . TrackinIn addition to exosome-mediated transfer of chemoresistance phenotypes between chemo-resistant and chemo-sensitive leukemic cells, exosomes can also transport cargo from AML cells to BMSCs that may contribute to the formation of a leukemia-supportive BM microenvironment . A recenin vitro and induce upregulation of both GRP78, a key chaperone protein involved in UPR, and spliced Xbp1, a transcription factor for chaperones and ER stress sensors  cell therapy in AML patients through interaction with activated NK-92 cells . More spin vitro and in vivo. Additionally, exosomal PD-L1 has been shown to act as a decoy, sequestering anti-PD-L1 checkpoint inhibitors and outcompeting the anti-PD-1 checkpoint blockade for binding sites on cytotoxic CD8+ T cells  . PD-L1 b T cells , 84. Sim T cells  (Butler,In vivo trafficking of EVs could also provide additional perspectives on how the chemoresistance phenotype is being transferred (per se, but rather to exploit our understanding of EV biology to identify cellular targets to overcome chemoresistance and achieve sustained long-term remissions. As an example, we previously have identified the role of miR-155, highly abundant in AML-EV, in the suppression of residual hematopoiesis in the AML BM (Recent research has uncovered numerous mechanisms through which EVs modulate proliferation, migration, and survival of malignant cells. While studies have implicated several specific proteins, cytokines, mRNAs, and miRNAs in the EV-mediated transfer of chemoresistance, the full spectrum of EV cargo involved in extrinsic chemoresistance remains to be fully defined. Additionally, the mechanisms through which many of these transferred miRNAs and proteins influence cell survival during chemotherapy are not fully understood. Investigation into leukemic stem cells and their role in transferring functional resistance factors similarly remains a potential focus for future AML EV research. nsferred . While pnsferred . Recent nsferred . It willnsferred . On the nsferred , 88. Thunsferred  represene AML BM . That ste AML BM . From a JN and PK conceived, wrote, and edited the manuscript. JB designed figures and wrote and edited the manuscript. D-WC wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite new insights in molecular features of leukemic cells and the availability of novel treatment approaches and drugs, acute myeloid leukemia (AML) remains a major clinical challenge. In fact, many patients with AML relapse after standard therapy and eventually die from progressive disease. The basic concept of leukemic stem cells (LSC) has been coined with the goal to decipher clonal architectures in various leukemia‐models and to develop curative drug therapies by eliminating LSC. Indeed, during the past few years, various immunotherapies have been tested in AML, and several of these therapies follow the strategy to eliminate relevant leukemic subclones by introducing LSC‐targeting antibodies or LSC‐targeting immune cells. These therapies include, among others, new generations of LSC‐eliminating antibody‐constructs, checkpoint‐targeting antibodies, bi‐specific antibodies, and CAR‐T or CAR‐NK cell‐based strategies. However, responses are often limited and/or transient which may be due to LSC resistance. Indeed, AML LSC exhibit multiple forms of resistance against various drugs and immunotherapies. An additional problems are treatment‐induced myelotoxicity and other side effects. The current article provides a short overview of immunological targets expressed on LSC in AML. Moreover, cell‐based therapies and immunotherapies tested in AML are discussed. Finally, the article provides an overview about LSC resistance and strategies to overcome resistance. Expression of cell surface target antigens on leukemic stem cells (LSC) in acute myeloid leukemia (AML) (left image) and on normal hematopoietic stem cells (right panel). Most of the clinically relevant surface target antigens are not only expressed on AML LSC but also in normal stem cells. In several instances, expression levels are higher on AML LSC than on normal stem cells providing a  therapeutic window. Only a few potential surface targets, such as CLL1 (CD371), are selectively (aberrantly) expressed on AML LSC but not on normal stem cells. Antibody‐based and cell‐based immunotherapies are emerging therapeutic approaches in applied hematology. However, although several attempts were made and several molecular markers have been identified, no curative immunotherapy is available in acute myeloid leukemia (AML). One explanation is that leukemic stem cells (LSC) are often resistant and are residing in several different fractions of the AML‐clone. This article discusses novel markers and targets expressed on LSC in AML, with emphasis on the potential value of these antigens in the context of antibody‐based and/or cell‐based therapies. Moreover, the article discusses various limitations and possible pitfalls of such therapies.1de novo AML, induction poly‐chemotherapy produces complete remission (CR), and many of these CR‐patients can be kept in continuous CR by introducing consolidation therapy. Notably, consolidation therapy is applied with the aim to eliminate most or all of the remaining leukemic (stem) cells after remission‐induction therapy.Acute myeloid leukemia (AML) is a life‐threatening malignancy characterized by an uncontrolled expansion of myeloid blast cells in the bone marrow (BM) and peripheral blood. Clinical presentation and courses in AML vary among patients, depending on the type of AML, molecular and immunological features of clonal cells, patient‐related variables such as age or comorbidities, and response to treatment.In the past 20 years, treatment of several AML variants has substantially improved which is mainly due to the availability of new (targeted) drugs, improved diagnostics, better selection of patients for various therapies , and advances in HSCT strategies.One strategy is to involve the immune system and to use targeted drugs and/or specific lymphoid killer cells that can attack and eliminate all AML cells . Another related strategy is to kill all AML cells that have a particular ability to propagate the malignancy for longer or even unlimited time‐periods.+/CD38−/Lin− subset of the AML clones.+/CD38+ subset or even in CD34‐negative AML cell subsets.The concept of leukemic stem cells (LSC) has been established to explain subclonal architectures and hierarchies in acute and chronic leukemias.In the past few years, the LSC concept has attracted more and more attention, and a rapidly expanding number of studies have discovered and validated distinct molecular targets and target‐pathways in these cells.In this article, target expression profiles of AML LSC are reviewed, and possibilities to design specific LSC‐eradicating therapies in AML are discussed, with special focus on antibody‐based and cell‐based therapy. In addition, the article reviews the multiple forms and mechanisms of LSC resistance and ways to overcome resistance.2in vivo, and (c) their capacity to survive well in extreme situations, toxic exposures, and total quiescence.+/CD38− AML cells but not by CD34+/CD38+ cells.+/CD38− fraction of AML cells. Later, it was found that the residual immune system of NOD/SCID mice can destroy CD38+ AML cells and that after blocking the residual immune system, also CD38+ AML LSC fractions can produce leukemic engraftment in the BM of NOD/SCID mice.+/CD38− and CD34+/CD38+ subsets of the malignant clone.+/CD38− and CD34+/CD38+ subfraction of leukemic cells.+/CD38− cells. The fact that AML LSC reside in multiple fractions of CD34+ cells, and sometimes (rarely) even in CD34‐negative cell populations in some AML variants,The three classic “stem cell features” of AML LSC are (a) their archaic self‐renewal capacity, (b) their long‐term disease‐propagating capability +/CD38− and most or all CD34+/CD38+ cells. Cell surface membrane structures that fulfill these criteria in most major AML variants and in most  patients are Siglec‐3 (CD33), Hermes (CD44), CD96, KIT (CD117), the interleukin‐3 receptor alpha‐chain (CD123), AC133 (CD133), FLT3 (CD135), and CXCR4 (CD184) .+/CD38−) stem cells do not express CLL‐1.So far, little is known about phenotypic properties of AML LSC. Based on LSC features described above, a surface marker or target can only be considered as being “expressed on AML LSC,” when it is detected on most or all CD346, KIT CD17, the i+ or CD34+/CD38− stem cells in AML (in subsets of patients) include, among others, CD93 and CD96.Additional surface membrane antigens expressed on CD34Finally, AML LSC display various immune checkpoint antigens, including CD47, PD‐L1 CD274), and the CTLA4 ligands CD80 and CD86  exhibited CD33.Gemtuzumab ozogamicin (GO) is a fully humanized CD33 antibody that is conjugated to the toxic drug calicheamicin through a chemical linker.In recent years, substantial efforts have been made to develop more potent CD33‐targeting antibody‐based drugs. Vadastuximab talirine (SGN‐CD33A) is a drug‐conjugate that contains a humanized anti‐CD33 antibody and a DNA‐targeting cytotoxic compound.388/IL‐3 fusion‐protein is such an agent.388 IL‐3[K116W], was developed by conjugating the toxin to a modified IL‐3 protein, resulting in increased binding‐affinity.388 IL‐3[K116W] is more effective than DT388 IL‐3 in killing AML blasts.in vivo in patients with AML remains uncertain. Efficacy of these agents presumably depends on expression of the IL‐3R on AML LSC. For both agents, however, the therapeutic window is small as normal stem cells also display (low amounts of) CD123. Another IL‐3‐toxin fusion protein is SL‐401 (tagraxofusp). In initial trials, this drug produced clinically meaningful responses in a smaller subset of patients with heavily pretreated AML.In the recent past, several attempts have been made to develop drugs attacking AML LSC through the IL‐3RA (CD123). An interesting approach is to conjugate the ligand, IL‐3, with a cytotoxic drug. The diphtheria toxin DTin vitro and in vivo on AML cells and AML LSC, while exerting less toxic effects on normal hematopoietic stem cells.Another approach to target the alpha chain of the human IL‐3R (CD123) is to develop antibodies that can bind to CD123 with high affinity and can thereby inhibit the binding and the effects of IL‐3 on AML cells.Other surface targets that have been considered in the context of AML and antibody‐mediated elimination of LSC include CD44, CD45, CD47, CD93, CD96, CD157, CD330f, and CLL‐1.131I‐, 213Bi‐, or 225Ac‐conjugated anti‐CD33 or CD45 antibodies, and 188Rhe‐labeled CD66 antibody.There are also a number of radiolabeled antibodies that have been considered for application in AML. Among these agents are 4Several immune checkpoint molecules, such as CD28, CD47, PD‐L1, PD‐L2, or TIM3, have been implicated in the resistance of malignant/leukemic (stem) cells in diverse hematopoietic malignancies. Moreover, it has been described that AML cells and AML LSC can express one or more of these immune checkpoint antigens Table .31, 4Overall, little is known about mechanisms contributing to the expression of PD‐L1 in AML LSC. Three principle mechanisms may be responsible: first, cytokines, especially IFN‐G and TNF‐A, are considered to induce/promote expression of this checkpoint antigen .41, 7In the recent past, molecular mechanisms contributing to cytokine‐induced expression of PD‐L1 on AML (stem) cells have been examined. In these investigations, the BRD4‐MYC axis and the JAK‐STAT pathway were described as major drivers of PD‐L1 expression.Other interesting checkpoint molecules in the AML context are CD47, the CTLA4 ligands CD80 and CD86 and TIM‐3.Another interesting checkpoint is TIM‐3. This antigen and its ligand, Galectin‐9, act in a constitute autocrine loop that appears to be essential for LSC survival in AML.5The so‐called bi‐specific antibodies are engineered compounds that consist of antigen recognition sites from two or more antibodies, thus providing binding‐interactions with two or multiple target antigens. These antibodies have certain advantages compared to conventional antibodies or monospecific toxin‐conjugates. For example, several of these bi‐specific antibody‐type drugs are able to recruit lymphoid killer cells onto the malignant target cells.There are several types of bi‐specific antibodies applied in clinical hematology.In the past few years, several efforts have been made to develop effective bi‐specific antibodies for the treatment of AML.Currently, the efficacy of these agents is evaluated in clinical trials: CD33‐directed bi‐specific antibodies examined in the AML context, are, among others, the CD33/CD3 antibody constructs AMG330 and AMG673, the tetravalent CD33/CD3 tandem‐diabody AMV564, and the humanized single‐chain bi‐specific CD33/CD3 construct GEM333.Bi‐specific antibodies directed against CD123 on AML LSC include, among others, the CD123/CD3 DART flotetuzumab, the CD123/CD3 bi‐specific IgG1 antibody JNJ‐63709178, and the CD123‐targeting bi‐specific antibody XmAb14045.6During the past 5 years, several brave attempts have been made to establish anti‐AML therapies by infusing immune cells expressing genetically adapted chimeric antigen receptors (CARs) (CAR‐T or CAR‐NK).However, several issues and limitations have to be taken into consideration when developing novel CAR‐T cell‐based therapies in AML. In particular, apart from cost‐related and logistic hurdles, CAR‐T cell treatment requires an extensive knowledge about the technique of CAR and about various practical and technical details. As a result, CAR‐T or CAR‐NK cell approaches can only be developed in highly specialized centers where AML is in focus of preclinical and clinical research. Second, a number of clinically relevant adverse events can occur during CAR‐T or CAR‐NK cell therapy in AML, such as a leukemia cell‐lysis syndrome, a CRS, sustained cytopenia (aplasia), and additional “on‐target but off‐leukemia” toxicities. One major problem is prolonged myelotoxicity especially when the CAR‐T approach will also eliminate normal stem cells.Another important issue in CAR‐based AML therapy is that current approaches are not addressing the diversity and the related clonal complexity of AML LSC, but rather focus on only one or two cell surface targets.7in vitro and to infuse these cells in AML patients together with donor stem cells during HSCT.In a majority of the patients with AML, the production and activity of T and NK cells are substantially impaired compared to T/NK cells in healthy individuals. Based on this notion, a number of therapeutic strategies have been coined with the idea to promote or even restore T cell and/or NK cell production and T/NK cell activity in AML patients and to mobilize these cells specifically against leukemic (stem) cells.As mentioned above, a number of attempts have been made to develop CAR‐NK cells against AML and AML LSC. However, it remains unknown whether CAR‐NK cells can better eliminate AML (stem) cells compared to CAR‐T cells.In recent years, a number of efforts have been performed to activate (prime) NK cells with stimulating cytokines and to transform these cells into optimized killer cells capable of attacking and eliminating AML LSC. Cytokine‐induced killer cells (CIK) are in general generated by exposing blood lymphocytes to IL‐1, IL‐2 and/or IL‐15. Usually CIK are heterogeneous populations of killer cells exhibiting both T‐cell (CD3) and NK (CD56) cell markers. These CIK have strong antileukemic effects and are able to introduce cytotoxicity against leukemic (stem) cells in an MHC‐restricted and an MHC‐unrestricted manner. A number of preclinical studies have been conducted with CIK in AML and several clinical phase I trials using CIK in refractory/relapsed AML patients have been performed.FLT3‐mutated AML in CR may also benefit from this therapy.A more conventional and historical way to boost antileukemic effects of immune cells is to induce NK‐ and T‐cell expansion and activation by infusing IL‐2. In earlier studies, however, no clinically meaningful effects of IL‐2 were observed in patients with AML. This may be due to the fact that immune cell responses against IL‐2 were suppressed by radical oxygen species (ROS) produced by accessory cells or immune cells. To overcome the ROS‐mediated suppression of immune cells, IL‐2 infusions were combined with histamine injections to block production of ROS.8One of the major problems in the treatment of AML is resistance of immature progenitor cells and stem cells against various therapies.The acquired form of resistance of AML LSC may or may not be responsive to various forms of immunotherapy or cell‐based therapies. For example, it may well be that antibody‐based or cell‐based immunotherapies can overcome mutation‐induced (acquired) resistance of AML LSC against tyrosine kinase inhibitors. However, it may also happen that mutation‐induced (acquired) resistance of AML LSC is associated with a “loss” of a certain surface target, such as CD33. Moreover, treatment of AML patients with CD33‐targeted antibodies or CAR‐T cells may well lead to a selection of CD33‐negative subclones and thus resistance  on normal stem cells. Another approach is to combine immunotherapy with stem cell transplanation. Finally, drugs with different mechanisms of actions and different target pathways can be selected, which often reduces (instead of enhancing) toxicity, especially when these drugs produce synergistic antineoplastic effects on AML cells, so that the concentration of the individual drugs can be reduced.9During the past 10 years, our knowledge about LSC, their target expression profiles, and their interactions with the stem cell niche and with certain immune cells, has increased substantially. At the same time, several new forms of cell therapy and other immunotherapies have been developed, and have recently been tested in patients with refractory or relapsed AML in clinical studies. These treatment approaches include new antibody‐based cytotoxic drug‐conjugates, administration of IL‐2 and histamine, application of CAR‐T or CAR‐NK cells, checkpoint‐specific therapies, and bi‐ or tri‐specific antibodies that recruit immune cells and/or cytokines to their target cells (LSC). In many instances, combinations of immunotherapies and intensive therapy are applied, and sometimes, drugs are multitargeted agents with the aim to achieve a broad effect on many or even most LSC subclones in AML. A similar combination effect is obtained when high‐dose chemotherapy and HSCT are combined with donor lymphocyte infusions, which is typically offered to patients with relapsed or refractory AML. However, in many cases, not all LSC can be eradicated, which is due to the multiple forms of LSC resistance. Another major problem is toxicity produced by stem cell‐directed immunotherapies in AML which is often due to the fact that normal stem cells display the same or almost the same targets compared to AML LSC. Therefore, current research is focusing on the development of new approaches to overcome LSC resistance and the toxicity issues with immunotherapy in AML. Whether these approaches will improve outcomes in AML therapy remains at present unknown.P.V. received a research grant from Celgene, a research grant from Pfizer and honoraria from Celgene and Pfizer. W.R.S. received honoraria from Celgene. The other authors declared no potential conflicts of interest.All authors contributed equally to this study by discussing potential immunological targets in AML and AML LSC, by drafting parts of the manuscript. P.V., W.R.S., W.R.: contribution in project design; K.B., I.S., D.S., D.I., H.H., Y.F., G.E.: contribution in stem cell phenotyping by flow cytometry. All authors approved the final version of the manuscript.AppendixS1: Supplementary InformationClick here for additional data file.


Signal-transducing adaptor protein (STAP) family members function as adaptor molecules and are involved in several events during immune responses. Notably however, the biological functions of STAP-1 in other cells are not known. We aimed to investigate the functions of STAP-1 in invariant natural killer T (iNKT) cells and iNKT cell-dependent hepatitis.We employed concanavalin A (Con A)-induced hepatitis and α-galactosylceramide -induced hepatitis mouse models, both are models of iNKT cell-dependent autoimmune hepatitis, and STAP-1 overexpressing 2E10 cells to investigate the role of STAP-1 in iNKT cell activation in vivo an in vitro, respectively.After Con A- or α-GalCer-injection, hepatocyte necrotic areas and plasma alanine aminotransferase elevation were more severe in STAP-1 knockout (S1KO) mice and milder in lymphocyte-specific STAP-1 transgenic (S1Tg) mice, as compared to wild-type (WT) mice. Two events that may be related to Con A-induced and/or α-GalCer-induced hepatitis were influenced by STAP-1 manipulation. One is that iNKT cell populations in the livers and spleens were increased in S1KO mice and were decreased in S1Tg mice. The other is that Con A-induced interleukin-4 and interferon-γ production was attenuated by STAP-1 overexpression. These effects of STAP-1 were confirmed using 2E10 cells overexpressing STAP-1 that showed impairment of interleukin-4 and interferon-γ production as well as phosphorylation of Akt and mitogen-activated protein kinases in response to Con A stimulation.These results conclude that STAP-1 regulates iNKT cell maintenance/activation, and is involved in the pathogenesis of autoimmune hepatitis. Autoimmune hepatitis is an inflammatory immune disease of the liver, and a worldwide health problem in humans. Because the only efficient therapeutic medication is glucocorticoid, patient quality of life is not high , 2. A be+ cells are crucial for the development of Con A-induced hepatitis [Traj18-deficient iNKT-lacking mice don’t exhibit liver injury after Con A treatment [Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell antigen receptor encoded by Vα14Jα18 gene segments . iNKT cereatment . In the reatment . Tiegs areatment . IL-12, Signal-transducing adaptor protein (STAP) family members function as adaptor molecules and are involved in several events during immune responses. We have previously investigated STAP-2, which is involved in various immune signaling events such as FcεRI-mediated mast cell/basophil activation , 14, intSTAP1 was identified in patients with autosomal dominant hypercholesterolemia [STAP-1 is a Tec tyrosine kinase binding partner that is predominantly expressed in the spleen and lymph nodes . Mouse Serolemia , 24 altherolemia , 26.Although several reports have suggested some functions of STAP-1, it is unknown whether STAP-1 is involved in the pathogenesis of immune diseases such as autoimmunity and allergy. In the present study we demonstrated that STAP-1 is required for the maintenance/activation of iNKT cells, and has a capacity to modify autoimmune hepatitis.TM 594-anti-mouse CD24 (clone: M1/69) and PE-anti-mouse NK1.1 (clone: PK136) mAbs were purchased from BioLegend . An anti-STAP-1 mAb (clone: S1/1) was generated in mice by immunization with recombinant STAP-1 as previously described [FITC-anti-mouse TCRβ (clone: H57-597), PerCP/Cy5.5-anti-mouse/human CD44 (clone: IM7), PE/Dazzleescribed .C57BL/6N mice were purchased from SANKYO LABO SERVICE CO. Inc. . A C57BL/6N background STAP-1 KO ES cells (EPD0583_5_G02) were purchased from European Conditional Mouse Mutagenesis Program. Human STAP-1 cDNA was inserted into the p1026x vector that consists of the murine lck proximal promoter, Ig intronic H chain enhancer Eμ, and a human growth hormone (hGH) gene cassette . The StaThe mice were intravenously injected with Con A  or α-GalCer  [Flowcytometric analysis was performed as previously described . FluoresMurine iNKT cell hybridoma, 2E10 , is cultWestern blot analysis was performed as previously described . NIH-3T3Statistical analysis was performed using GraphPad Prism 6.02. Mann-Whitney U-test was employed. Data were considered significant at p<0.05. Data were shown mean + SEM.Endogenous STAP-1 expression was tested in splenic and thymic tissues from C57BL/6 mice, and in cell lines including NIH-3T3 (fibroblasts), Ba/F3 (pro B cells), and 2E10 (iNKT cells). It was detected in the murine splenic and thymic tissues and in Ba/F3 cells and 2E10 cells, but not in NIH-3T3 cells . To inveTo investigate whether STAP-1 is involved in the pathogenesis of autoimmune hepatitis, Con A-induced hepatitis—a murine model of autoimmune hepatitis—was induced in S1KO, S1Tg, and WT mice. Liver tissues from Con A-treated S1KO mice exhibited more severe necrosis than those from WT mice, whereas Con A-treated S1Tg mice exhibited less liver injury than WT mice . The difIL-4 and IFN-γ are important cytokines to mediate murine Con A-induced hepatitis , 30, thuα-GalCer is a stimulator, which is more restricted to iNKT than Con A. To investigate whether STAP-1 is a critical adaptor protein to induce iNKT cell-mediated hepatitis, the effects of STAP-1 on α-GalCer-induced hepatitis were assessed . To conf+/T cell receptor (TCR)β+ iNKT cells tended to be increased in S1KO mice, but they were significantly reduced in S1Tg mice  were generated. Endogenous STAP-1 (~37 kDa) was expressed in Mock vector-transfected 2E10 cells (Mock 2E10 cells) . STAP-1 in vivo and in vitro; in addition, STAP-1 is likely to be a key player in the pathogenesis of autoimmune hepatitis.In summary, STAP-1 regulates Con A-induced and/or α-GalCer-induced hepatitis in which iNKT cells have important roles. STAP-1 is involved in at least two events during the pathological process. One is iNKT cell homeostasis in the liver and spleen, and the other is the inhibition of Con A-mediated signals, which leads to reduced IL-4 and/or IFN-γ production. Present data suggest new functions of STAP-1 in vivo. One report suggests that leukocyte cell-derived chemotaxin 2 (LECT2) is an important cytokine in the regulation of iNKT cell homeostasis in the liver. LECT2-deficient mice are known to exhibit a reduced number of iNKT cells in the liver, but not the spleen [Con A-induced hepatitis is an experimental model for autoimmune hepatitis, and iNKT cells are required for the development of the type of hepatitis in mice . In thise spleen . The difCD137, also known as 4-1BB, is a member of the tumor necrosis factor receptor superfamily (TNFRSF) and is expressed on immune cells such as T cells and NK cells. iNKT cells also express CD137, and CD137 signaling is crucial for maintaining iNKT cell numbers in the liver and spleen . Our preData derived from 2E10 cells overexpressing STAP-1 indicated direct inhibition of signals after Con A stimulation. Although details pertaining to the mechanisms involved remain to be determined, ITK may be a candidate to be regulated by STAP-1 in iNKT cells. ITK is a member of Tec family of tyrosine kinases, and our previous research indicates that STAP-1 binds to another Tec family member, Tec . ITK is S1 File(PDF)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.