However, its daughter phase,the brownmillerite SrCoO2.5 (BM-SCO),is insulating and an antiferromagnet. Moreover, BM-SCO exhibits oxygen vacancy channels (OVCs) that in thin films canbe oriented either horizontally (H-SCO) or vertically(V-SCO) to the film’s surface. To date, theorientation of these OVCs has been manipulated by control of the thinfilm deposition parameters or by using a substrate-induced strain.Here, we present a method to electrically control the OVC orderingin thin layers via ionic liquid gating (ILG). We show that H-SCO  can be convertedto P-SCO  and subsequentlyto V-SCO (AFI) by the insertion and subtraction ofoxygen throughout thick films via ILG. Moreover, these processes areindependent of substrate-induced strain which favors formation of H-SCO in the as-deposited film. The electric-field controlof the OVC channels is a path toward the creation of oxitronic devices.Oxygen defects andtheir atomic arrangements play a significantrole in the physical properties of many transition metal oxides. Theexemplary perovskite SrCoO An importantexample are the brownmillerite (BM) phases that areformed from the parent perovskite phase by the removal of oxygen inalternate layers: representative examples are SrCoO2.5,18 BaInO2.5,19 Lax1–SrxCoO3-δ,26 SrFeO2.5,31 SrFex1–CoxO2.5,31 and CaFeO2.5.35 In these phases, oxygen vacancy channels (OVCs) areformed within the oxygen deficient layers. The OVCs can provide pathsfor fast ionic diffusion11 or storage ofions,33 leading to applications in fuel cells andrechargeable batteries. In addition, the OVCs affect the resistiveswitching response in memristors10 and strongly affectmagnetic anisotropy.8Oxygen vacancies playan important role in determining the physicalproperties of many transition-metal oxides (TMO). The oxygen vacanciescan give rise to phase transitions, for example, between metallicand insulating states, and can significantly modify the electrical,magnetic, and optical properties.31 by the deposition conditions of the filmgrowth,30 or by using an appropriate capping layer.20 However, all these methods are passive and cannotbe employed to control the OVCs in films after they have been formed.Here we show a means of electrically controlling the formation ofthe OVCs in thin layers of the exemplary oxide SrCoOx using ionic liquid gating (ILG) which has been shown to modifythe properties of thin films of several TMO by the electric fieldinduced removal or insertion of oxygen into the film lattice.5 The phase transformation between brownmillerite and perovskite isenabled by applying an electric-field even at room temperature owingto the low oxygen vacancy formation energy and the high oxygen vacancydiffusivity of such systems.25In thin films, the OVCscan be formed either parallel or perpendicularto the substrate surface and thereby strongly influence the propertiesof the films. The OVC orientation can be affected by applying strainin thin films by suitable choice of the substrate on which the filmis deposited,x is a fascinating material. Whereasthe parent perovskite SrCoO3-δ (P-SCO) is a ferromagnetic metal (FM), brownmillerite SrCoO2.5 (BM-SCO) is an antiferromagnetic insulator (AFI).It has previously been shown that ILG of SrCoOx leads to a reversible transition between the BM-SCO and P-SCO phases.16 Here we demonstratethat BM-SrCoO2.5 with horizontal OVCs(H-SCO) can be transformed to the perovskite phase(P-SCO) that has no OVCs, and that P-SCO can be transformed to BM-SCO with vertical OVCs(V-SCO) by ILG. V-SCO can transformback to P-SCO but not to H-SCO.Notably, these transformations are shown to be independent of straininduced in SrCoOx thin films by growingthem on three distinct substrate materials. Our findings provide amethod for the control of the OVCs in thin oxide films via ILG.SrCoOBM-SCO and P-SCO were grown on SrTiO3 (STO)(001) substratesusing pulsed laser deposition (PLD). (La0.18Sr0.82)(Al0.59Ta0.41)O3 (LSAT) and LaAlO3 (LAO) (001) substrates were also used to investigate straineffects, as discussed later. The films were then gated by ILG at differentgate voltages. Details are given in Supporting Information (SI) Figure S1 and the SI Figure S2 shows X-ray diffraction(XRD) and resistivity measurements at room temperature for BM-SCO and P-SCO films after gating ata sequence of gate voltages for 1.5 h at each voltage. Bulk BM-SCO (related to H-type SCO as discussedbelow) crystallizes in an orthorhombic unit cell with the space group Ima2, characterized by lattice parameters, a = 5.470 Å, b = 5.574 Å, and c = 15.745 Å,36 while bulk P-SCO has a cubic perovskite unit cell with the space group Pm3̅m, and a = 3.836Å.37 Before ILG, BM-SCO films grown on STO (001) show distinct peaks indexed by (002),(006), (0010), that are consistent with the body-centered latticerelated to the alternate stacking of octahedral CoO6 andoxygen deficient tetrahedral CoO4 layers along the [001]axis. The unit cell of the BM-SCO film can be consideredas an approximate SI). The observation of the (00L) reflections in the specular θ-2θscans indicates the presence of H-SCO. By applyingnegative gate voltages exceeding a magnitude of 1.5 V (U> |−1.5V|), the H-SCO structure is transformed to P-SCO, which goes in parallel with a significant decreasein resistivity. On the other hand, we observe an increase of the resistivityif P-SCO is gated at positive gate voltages. Aftergating above 1 V, we observe reflections which are related to V-type BM-SCO (110) and (220) peaks, whilethe reflections related to H-SCO are not evident.The BM-SCO (004) and (110) peak positions are locatedat nearly the same 2θ-angle of approximately 23°. Similarly,the P-type (008) and the V-type(220) peak positions are close near 2θ ∼46°. Thismakes it difficult to immediately recognize any phase change. However,because H-SCO reflections along (00L) are not observed,the peaks at 2θ ∼23° and ∼46° must correspondto V-type BM-SCO (110) and (220),respectively, thus indicating the ILG induced formation of V-SCO. To confirm this transformation we carried out nonspecularazimuthal phi (φ) scans . Considering that the V-type BM-SCO film is (110) oriented, the film surface was tilted to a polarchi (χ) angle of approximately 56°, which corresponds tothe angle between the (110) plane parallel to the film surface andthe (116) plane. The set of {116} reflections is observed versus φfollowing an in-plane 4-fold symmetry . Thus, these nonspecular scans confirm that ILG carriedout at positive gate voltage transforms P-SCO to V-SCO. The φ-scan of the H-SCO filmreveals 4-fold symmetric BM-SCO (114) reflectionswhich are offset from the STO (111) peaks by 45° . This relative orientation betweenfilm and substrate leads to the smallest lattice mismatch that willbe described later. On the other hand, P-SCO growsby a simple cube-on-cube relation on STO (001) as shown SI Figure S3c. For the V-SCOfilm, we hereafter reindex the BM-SCO (110) and (220)peaks as V-SCO (001) and (002) peaks, respectively.Cross-sectional scanning transmission electron microscopy (STEM)-highangle annular dark field (HAADF) measurements are conducted to confirmthe structure and OVC orientations of the three films . H-SCO films showhorizontal OVCs in which the Co ions within the OVCs are all locatedin tetrahedral sites. The well-known dimerization14 of the Co ions within the OVCs are clearly seen in theSTEM images taken along the [100] zone axis . This means that within the oxygen vacancy planesthere are two distinct directions which are oriented at an angle of90 deg relative to each other with different oxygen ion vacancy diffusivities.38 The V-SCO film reveals verticallyoriented OVCs but the Co–Co dimerization cannot be seen alongthis zone axis. The P-SCO shows a conventional perovskitestructure without any OVCs.Epitaxial, 30 nm thick, films of x inducedby ILG. H-SCO transforms to P-SCOat negative gate voltage. Furthermore, positive gate voltage transforms P-SCO to V-SCO, which can then be reversiblytransformed back to P-SCO by negative gate voltage. Thus, the orientation ofthe OVCs can be controlled by an electric field induced at the ILG/oxideinterface, which also goes in parallel with significant changes ofthe physical properties of the film. As shown in M-H) collected at 100 K for P-SCO indicates ferromagnetism (FM), while the loops for H-SCO and V-SCO are consistent with theknown antiferromagnetic (AFM) ground state of BM-SCO.36 Note that P-SCO transformsonly to V-SCO but not to H-SCO nomatter what the magnitude of the gate voltage is applied .ltageSI S5. Thus,x phases discussed abovehave distinct physical properties. Temperature dependent magnetizationversus temperature (M-T) curves recorded at 1000Oe (M-H) curvesrecorded at 100 K, mentioned above, show clear evidence for ferromagnetismonly in P-SCO. The saturation magnetization (23 emucm–3 or 0.15 μB/Co) determinedfor the P-SCO film is smaller than that of bulk P-SCO.37 It is challenging tomake a fully oxidized SrCoO3 thin film because the valencestates of Co2+ and Co3+ are more stable thanCo4+.14 Thus, the smaller magnetizationis likely due to the incompletely oxidized state of the film. thatis, SrCoO3-δ.14 Temperature-dependent resistivity curves  measurements were carried out to distinguish the chemical statesof Co and O in the three phases . XPS spectra reveal that the Co 2p core levelsof P-SCO have a higher binding energy than thoseof H- and V-SCO, which is attributedto the higher degree of covalent character of the Co–O bondin P-SCO.40 O 1s XPS spectra show a distinct peak at the lower binding energy sidein the case of H-SCO and V-SCO whichoriginate from the oxygen deficient CoO4 tetrahedral layers,which is therefore missing in the case of P-SCO. L2,3 edge for the three phases at 2 K. The absorptionspectra of H- and V-SCO are characterizedby a similar multiplet structure at the L3 edge, while a small intensity difference between them might be dueto the different OVC orientations between H- and V-SCO relative to the incident X-ray beam. Moreover, thepeak shift (observed for both L3 and L2 peaks) toward higher energy in the P-SCO spectrum indicates an increased valence state of Co14 as compared to H- and V-SCO.The three SrCoO 1000Oe 2a andmay curves 2c show ahases SI S6. XPS sL2,3 edge in zero field after applying an in-plane magneticfield of 6 T. Only for P-SCO, a finite signal isobserved related to its FM order, while for the AFM phases H- and V-SCO, no XMCD signal is observedwithin the experimental accuracy. Normalized O K-edgeXAS spectra  further confirmthat a ferromagnetic hysteresis loop exists only for P-SCO. In summary, these studies reveal that the electronic, magnetic,and chemical properties between H- and V-SCO are similar, while they are distinctly different in the caseof P-SCO.To complement these data, X-raymagnetic circular dichroism (XMCD)measurements were carried out at 2 K.  spectra 2f exhibiP-SCO versus 3.00 for STO). In the case of V-SCOthere is almost no relaxation (the peaks nearly coincide) while for H-SCO the c-lattice parameter is almost four times largerthan that of STO so that this reflection corresponds to L = 12.The mutual crystallographic relationbetween the STO substrateand the film lattice was examined using X-ray diffraction by recordingreciprocal space maps (RSMs) 2g of theH- and V-SCO structure was carried out by using a GaJet X-ray source(λ = 1.3414 Å) and a six-circle X-ray diffractometer designedfor the study of thin films and surfaces. Structure refinements werecarried out using 33 and 22 symmetry independent reflections collectedfor H- and V-SCO, respectively.The detailed analysis is described in the SI. The H-SCO film can be viewed as an in-plane V-SCO film correspondsto a (2 × 1) superstructure We find that the structure of H- and V-SCO can be mapped to the spacegroups Ima2 and Pmmm, respectively.The lattice constants of H- and V-SCO films grown on STO (001) substrates are given in SI Table S1. SI Figure S8 shows a schematic view of the H-SCO structure,which is identical to the reported brownmillerite phase: the atomiccoordinates agree to within 0.02–0.05 lattice units. For V-SCO, which is shown in SI Figure S9, we find an oxygen vacancy located at the sites labeled (1) and(2). These sites are approximately occupied by 50% each, correspondingto two (coherent) domains in which either site (1) or site (2) hasa vacancy. This is due to the fact that beginning with the parent P-SCO (1 × 1) phase there are two symmetry equivalentsites to create a vacancy both leading to the (2 × 1) superstructure.In addition there are two rotational domains which are related toeach other by a 90° rotation, resulting in a (2 × 1) anda (1 × 2) superstructure domain which, however, do not interferein reciprocal space. In addition, there is a small (∼0.01 latticeunits) relaxation of the Sr ions along the a-axis.A more in-depth analysis of the V-SCO,41 we have selected compressively strained filmsto investigate the ILG induced OVC ordering beginning with as deposited H-SCO films. All H-SCO films are foundto follow the same ILG induced OVC ordering sequence, namely from H-SCO, to P-SCO, to V-SCOregardless of strain  to −3% (εa of V-SCO/LAO). Thus, the OVC ordering induced by ILG occursat the expense of the large strain. The time-dependent changes ofsource-drain current (ISD) during ILGand the subsequent phase change of SCOx films on LSAT (001) are further described in SI Figure S10.The ILG controlled OVC ordering was further investigated by employingthree different, compressively strained films grown on an STO (001),LSAT (001), and LAO (001) substrate see 3. Since f strain 3a-3c. PsP-SCO is further studiedby an annealing treatment. P-SCO films grown on thethree substrates were annealed at 750 °C in an oxygen pressure(pO2) of 10–2 Torr for30 min. P-SCO films are transformed to H-SCO which is at variance with the ILG induced phase transformation.The phase change via ILG can be accounted for by the formation ofan electric double layer (EDL) at the ionic liquid/oxide interfaceand the subsequent electric field induced migration of oxygen perpendicularto the surface.42 Thus, as depicted in P-SCO films leads to the migration ofoxygen ions along a vertical pathway, resulting in vertically orientedOVCs. In contrast to our results, some other BM systemssuch as La0.5Sr0.5CoO3-δ25 and SrFe0.5Co0.5O3-δ32 have shownhorizontal OVC ordering from the gating of the perovskite phase. Thesubstitution of Co by Fe and Sr by La significantly changes the filmproperties such as chemical stability, magnetism, and OVC ordering.27 For example, SrFeO2.5 films grown on STO (001) were foundto develop vertical or horizontal OVC ordering just by changing thedeposition conditions.27 Thus, dependingon the chemical composition of the BM phase and thegating conditions, distinctly different behavior of the OVC orderingis expected. Their investigation is subject to further studies.The phase transformation of H-SCO structure can be transformedinto the V-SCO phase by a sequence of two ILG steps and the (008) reflection at 2θ∼58°and 45.8°, respectively. Upon gating with a positive gate voltageduring the H-SCO to P-SCO transformationthese peaks gradually disappear within about 20–25 min whilethe (002) peak of the P-type film appears after about25 min with almost no time overlap. Subsequently, negative gate voltagebiasing leads to the formation of the V-SCO phaseas concluded by the appearance of the V-SCO (002)peak and the simultaneous disappearance of the P-SCO(002) reflection. Also, there is no trace of the H-SCO (0010) reflection.Films that have a LG steps5b. Finalx irrespectiveof strain in the film that would otherwise favor horizontally orderedOVCs. This concept can be used for a wide range of diverse deviceapplications.In summary, we have demonstrateda method for controlling the orientationof the oxygen vacancy channels in thin oxide films, here of the exemplarytransition metal oxide SrCoO2 wereused. H-SCO films were grown at 770 °C in anoxygen pressure of 10 mT. P-SCO films were grownat 770 °C using an oxygen pressure (pO2) of 300 mT, and then postannealed at 770 °C in 500 Torr (pO2) for 30 min. To carry out ILG of the films,the samples were cut into small chiplets, each with an area of ∼3.3× 5 mm2, using a diamond scriber.Thirty nm thick epitaxial SCO filmswere grown on several substrates by pulsed laser deposition  using a 248 nm KrF excimer laser source. LAO, LSAT, andSTO (001) substrates with a size of 10 × 10 mm2)that was placed adjacent to the chiplet . Both the chiplet and Au foil were covered with an ionicliquid, DEME-TFSI  ammonium bis(trifluoromethanesulfonyl)imide).After gating, the liquid was removed by placing the sample in a beakerof acetone followed by isopropyl alcohol. For the kinetics measurements,source-drain current (ISD) was measuredduring gating of SCO/LSAT .ILG was carriedout in a polytetrafluoroethylene (PTFE) boat in which the gate electrodewas formed from a 0.5 mm thick Au foil . Then, electrical contact pads were formed from Au (70nm)/Ru (5 nm) bilayers by a lift-off technique. The Ru and Au weredeposited by ion beam sputtering (SCIA coat 200).Standard photolithographic methodswere used to fabricate devices for transport measurements. Channels,65 × 30 μmInsitu XRD measurements were carried out using a portion ofan H-SCO film grown on LSAT (001). The chiplet usedhad an area of 5 × 5 mm2. A channel with an area of2 × 2 mm2 was defined and then Au (70 nm)/Ru (5 nm)electrodes were formed. A gate electrode with an area of 0.8 ×2 mm2 was formed on the chiplet next to the device itself. The device was attachedto the sample holder using double-side tape. After placing the ionicliquid on the device surface, a Kapton film was attached to reduceits thickness.in situ XRD, and RSM were performed using a Bruker D8 Discover X-ray diffractometerand Cu–Kα radiation. A GaJet X-ray source (λ =1.3414 Å) XRD was used to analyze the detailed structure of the H- and V-SCO thin films as described inthe SI. STEM-HADDF measurements were carriedout in a FEI Titan 80–300 transmission electron microscopewith an electron beam energy of 300 kV. X-ray absorption spectroscopy(XAS) and X-ray magnetic circular dichroism (XMCD) measurements wereconducted at the vector cryomagnet XMCD end station of the BOREASbeamline at the ALBA synchrotron source.43 The measurements were carried out at a base pressure of <1 ×10–10 mbar and under an applied magnetic field upto 6 T along the incident beam direction. Co L2,3 and O K edge spectra were collected atgrazing incidence  at 2 K, using total electronyield (TEY) detection. Nearly 100% circularly polarized left and rightX-rays were formed using an elliptically polarizing undulator. Additionally,element-specific hysteresis loops were performed by recording theTEY signal at the maximum value of the XMCD at the L3 edge and at a pre-edge energy for normalization purposeswhile sweeping the magnetic field, for both X-ray polarizations. X-rayphotoelectron spectroscopy (XPS) measurements were made using a K-AlphaThermo Scientific instrument. The film surface was gently cleanedby an Ar cluster ion etching prior to the measurement.Temperature andmagnetic field dependentmagnetization was measured using a MPMS3 SQUID (Superconducting QuantumInterference Device) magnetometer. Transport measurements were carriedout in a physical property measurement system (Quantum Design). Thegate voltage was applied using a Keithley 2450A source meter. Thefilm resistance was measured using a constant current of 500 nA (Keithley6221 current source), and the voltage was measured using a Keithley2182A nanovoltmeter. θ–2θ scans,


Importantly, anti-CCR8 CAR T cells exhibited antitumor effects on ATLL- and other CCR8-expressing T-ALL cells in vitro and in vivo, and prolonged the survival of ATLL and Jurkat tumor-bearing mouse models. In conclusion, these collective results show that anti-CCR8 CAR T cells possess strong antitumor activity and represent a promising therapeutic approach for ATLL and CCR8+ tumors.Chimeric antigen receptor (CAR) T cells have been successfully used in the therapy of B cell leukemia and lymphoma, but still have many challenges in their use for treating T cell malignancies, such as the lack of unique tumor antigens, their limitation of T cell expansion, and the need for third party donors or genome editing. Therefore, we need to find novel targets for CAR T cell therapy to overcome these challenges. Here, we found that both adult T-cell leukemia/lymphoma (ATLL) patients and ATLL cells had increased CCR8 expression but did not express CD7. Moreover, targeting CCR8 in T cells did not impair T cell expansion  Although chimeric antigen receptor (CAR) T cell therapies lead to high clinical response rates in patients with certain B cell malignancies , 2, theiAdult T-cell leukemia/lymphoma (ATLL) and peripheral T-cell lymphoma (PTCL) are major subtypes of T-cell lymphoma . ATLL is+ T cells, myeloid cells, γδT cells, the bulk of CD4+ Tconv cells, and Treg cells found in peripheral blood did not express CCR8 , is induced by the CC chemokine CCL1/I309 , 14. Peress CCR8 . CCR8 coess CCR8 , and bloess CCR8 . CCR8 isess CCR8 . Therefohttps://www.ncbi.nlm.nih.gov/geo/), and the download data format is MINIML. Box plots are drawn by boxplot; PCA graphs are drawn by R software package ggord; The box plot is implemented by the R software package ggplot2; the heat map is displayed by the R software package pheatmap  used is from GEO database  PCT/JP2019/051603) and anti-CCR8 (19) (PCT/JP2019/051603) CAR vectors incorporating CD28, TLR2 19/051603 and CD3ζ6 cells/ml for two days in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ml recombinant human IL-2, 10 mM HEPES, 2 mM glutamine and 1% penicillin/streptomycin. On Day 2 post activation, T cells were transduced with CAR vector lentiviral supernatants in the presence of 8 μg/mL polybrene at a multiplicity of infection (MOI) of 2.0 . Twelve hours after transduction, T cells were cultured in fresh medium containing IL-2 (300 U/mL); subsequently, fresh medium was added every 3 days to maintain the cell density at approximately 1 × 106 cells/ml. T cells were transduced with the CAR-expressing lentiviral vectors for 24 h. The healthy PBMC donors provided informed consent for the use of their samples for research purposes, and all procedures were approved by the Research Ethics Board of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (GIBH).Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult donors using Lymphoprep . T cells were negatively selected from PBMCs using a MACS Pan T Cell Isolation Kit  and activated using microbeads coated with anti-human CD3, anti-human CD2 and anti-human CD28 antibodies  at a bead:cell ratio of 1:2 and a density of 2.5×10HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) . Cell lines such as Jurkat/Jurkat-GL (T-acute lymphoblastic leukemia), Molt-4 (T-acute lymphoblastic leukemia), MT-4/MT-4-GL (adult T-cell leukemia/lymphoma), and C8166/C8166-GL (adult T-cell leukemia/lymphoma), were maintained in RPMI-1640 medium. The medium was supplemented with 10% heat-inactivated FBS , 10 mM HEPES, 2 mM glutamine  and 1% penicillin/streptomycin . All cells were cultured at 37°C in an atmosphere of 5% carbon dioxide.6 Tumor cells transduced with 10 ml GL-lentiviral particles for 12 h, and the GFP percentage of GL-tumor cells were detected through flow cytometry for 48 h. GL-tumor cells were sort by FACS Aria. The purity of GL-tumor cells were >95% for killing assay.The GFP-2A-Luciferase (GL) vector contains EGFP, Luciferase and 2A sequence and clones by Sangon Biotech company . GL-Lentiviral particles were produced in HEK-293T cells following polyethyleneimine -mediated transfection with the pWPXLd-based transfer plasmid and the packaging and envelope plasmids psPAX2 and pMD2.G. Lentivirus-containing supernatant was harvested at 24, 48, and 72 h post transfection and filtered through a 0.22-µm filter. 1 × 10Flow cytometry was performed on a Fortessa cytometer , and data were analyzed using FlowJo software . The antibodies used, including anti-human CD3-PE (UCHT1), anti-human CD4-APC-Cy7 (OKT4), anti-human CD8-PE_Cy7 (OKT8), anti-human CCR8-APC (SA214G2), and anti-human CD7-FITC (4H9/CD7), were purchased from Biolegend . Staining was performed on ice for 30 minutes, and cells were then washed with PBS containing 2% FBS before cytometric analysis. For intracellular staining, cells were fixed and permeabilized with a Foxp3/Transcription factor staining kit , followed by staining with transcription factor-specific antibodies, such as Tbet, Gata3 and Foxp3. Cells were gently vortexed and were then incubated in the dark for 30 minutes at room temperature. Afterward, the cells were washed once more with cold flow buffer and were then analyzed immediately.MT-4-GL, C8166-GL and Jurkat-GL target cells were incubated with 1928z, C1028z or C1928z T cells at the indicated ratio in triplicate wells of U-bottom 96-well plates. Target cell viability was monitored 18 hours later by adding 100 µl/well of the substrate D-luciferin   at 150 µg/mL. Background luminescence was negligible . The percentage of viable target cells (%) was calculated as / ×100, and the percentage of cytotoxicity was calculated as 100 – percentage of viable target cells.T cells were cocultured with target cells at an E:T ratio of 4:1 for 24 hours, and supernatants were analyzed for cytokine release by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ protocols. ELISA kits for IFN-γ and Granzyme-B were purchased from Thermo Fisher Scientific Inc., USA.6 transduced human T (GFP+ or CAR+) cells in 200 μL of PBS were adoptively transferred into tumor-bearing mice systemically by tail vein injection. Peripheral blood was obtained by retro-orbital bleeding. Body weight was measured every 2–3 days as indicated. Tumors were measured every 3 days with a caliper. Tumor volume was calculated using the following formula: (length×width2)/2. In vivo whole-body imaging of luciferase-labeled cells was performed using a cooled CCD camera system . D-luciferin Firefly, potassium salt was injected at 75 mg/kg. Mice were imaged 5 minutes after injection of the substrate. Quantification of the total and average emission was performed using Living Image software.Animal experiments were performed in the Laboratory Animal Center of GIBH, and all animal procedures were approved by the Animal Welfare Committee of GIBH. All protocols were approved by the relevant Institutional Animal Care and Use Committee (IACUC). NSI mice  were maiStatistical significance was determined using Student’s t test (two groups) or ANOVA with Tukey’s multiple comparison test (three or more groups). All statistical analyses were performed using Prism software, version 7.0 . The gene distribution of GEO database was analyzed using wilcox tests. Kaplan-Meier survival curves of vivo experiments were analyzed using log-rank. P values < 0.05 were considered statistically significant, and the following annotations were used: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. For assessment of differential gene expression, a minimum fold change of 2 was used, and a false discovery rate-corrected P value of < 0.05  was considered significant .in vitro   C1028z and anti-CCR8 (19D7) C1928z, and anti-CD19 CAR (1928z) served as negative control, which contain the scFv, the human CD28 transmembrane domain (CD28TM) and endodomain, the human CD3ζ T cell activating domain, a human TLR2 domain (T2) and EGFP; these vectors were introduced into human T cells through lentiviral transduction  patients have an extremely poor prognosis that cannot be prolonged through chemotherapy alone. Here, we show that activated human T cells that express the CCR8 CAR can specifically recognize and kill ATLL cells in vitro , the National Natural Science Foundation of China , The Youth Innovation Promotion Association of the Chinese Academy of Sciences (No. 2020351 to ZJ), the Guangdong Provincial Significant New Drugs Development , the Guangzhou Science and Technology Plan Project (No. 201907010042 to PTL), 2020B1212060052, the Frontier Research Program of the Guangzhou Regenerative Medicine and Health Guangdong Laboratory (No. 2018GZR110105003 to PTL), the Science and Technology Planning Project of Guangdong Province, China (2020B1212060052), the Science and Technology Program of Guangzhou, China (No. 202002020083 to XL), the Open Project of the State Key Laboratory of Respiratory Disease (No. SKLRD-OP-202002 to ZZ), the University Grants Committee/Research Grants Council of Hong Kong (Project No. AoE/M-401/20), and the Innovation and Technology Fund (ITF) from the Hong Kong SAR government, the Youth Talent Promotion project of Guangzhou Association for Science and Technology(No. X20210201015 to LY).In vivo Biomedicine Ltd. and Guangdong Zhaotai Cell Biology Technology Ltd.PTL is a scientific founder of GZI and GZCB and has equity in GZI and GZCB. Author ZT was employed by Guangdong Zhaotai The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The effect of the autoantibodies on the formation, activity and FH-mediated decay of the C3 convertase in solid phase convertase assays was determined. In order to investigate the effect of the autoantibodies on complement functions, hemolysis assays and fluid phase complement activation assays were performed. The autoantibodies partially inhibited the complement-mediated hemolysis of rabbit red blood cells, inhibited the activity of the solid phase C3-convertase and C3 and C5b-9 deposition on complement activating surfaces. In summary, in ACPA-positive RA patients we identified FB autoantibodies. The characterized FB autoantibodies did not enhance complement activation, rather, they had inhibitory effect on complement. These results support the involvement of the complement system in the pathomechanism of RA and raise the possibility that protective autoantibodies may be generated in some patients against the alternative pathway C3 convertase. However, further analyses are needed to assess the exact role of such autoantibodies.Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder affecting the joints. Many patients carry anti-citrullinated protein autoantibodies (ACPA). Overactivation of the complement system seems to be part of the pathogenesis of RA, and autoantibodies against the pathway initiators C1q and MBL, and the regulator of the complement alternative pathway, factor H (FH), were previously reported. Our aim was to analyze the presence and role of autoantibodies against complement proteins in a Hungarian RA cohort. To this end, serum samples of 97 ACPA-positive RA patients and 117 healthy controls were analyzed for autoantibodies against FH, factor B (FB), C3b, C3-convertase (C3bBbP), C1q, MBL and factor I. In this cohort, we did not detect any patient with FH autoantibodies but detected C1q autoantibodies in four patients, MBL autoantibodies in two patients and FB autoantibodies in five patients. Since the latter autoantibodies were previously reported in patients with kidney diseases but not in RA, we set out to further characterize such FB autoantibodies. The isotypes of the analyzed autoantibodies were IgG2, IgG3, IgGκ, IgGλ and their binding site was localized in the Bb part of FB. We detected  Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder targeting typically the peripheral joints of the hands and feet, leading to persistent inflammation. With time it may cause the formation of a new tissue with infiltrating inflammatory immune cells called pannus and the destruction of the bones and cartilage which may give rise to deformities , 2. RA aMost of the patients develop rheumatoid factor or anti-modified protein antibodies. The classical serological marker of RA was rheumatoid factor, an autoantibody targeting the Fc region of IgG. It has relatively low specificity as it can be found in other pathological conditions and healthy individuals , 13, 14.2O)). Thus, factor B (FB) bound to C3(H2O) and cleaved by factor D (FD), forms the fluid phase C3 convertase of the alternative pathway, C3(H2O)Bb. This in turn cleaves C3, generating C3b which can covalently bind to target surfaces and together with FB  and the stabilizing properdin forms the C3 convertase of the alternative pathway C3bBbP. The activation of the cascades leads to the generation of small anaphylatoxins  and larger fragments which opsonize the target . FD (#A409), properdin (#A412), mouse anti-C5b-9 (#A239), anti-FB (Bb) (#A227), anti-FB (Ba) (#A225) and C3a EIA kit (#A032) were from Quidel . Purified human IgG (#I2511), human serum albumin (#A3782), human alpha-1 antitrypsin (#SRP6312), antibodies against C1q (#234390), human IgG (#A6029), IgM (#A0420), IgA (#A0295), IgG1 , IgG2 , IgG3 , IgG4 , IgGκ , IgGλ , and avidin-HRP (#A7419) were purchased from Merck. Mouse IgG1  was obtained from BioLegend . MBL (#9086-MB) and biotin-conjugated goat anti-MBL (#BAF2307) were from R&D Systems , HRP-conjugated goat anti-mouse (#P0447), rabbit anti-goat (#P0449) and swine anti-rabbit (#P0217) antibodies were from DAKO . Goat anti-C3 F(ab’)2 (#55062) and HRP-conjugated goat anti-C3 F(ab’)2 (#55237) were from MP Biomedicals .Complement proteins FH (#341274), FB (#341262), C3b (#204860), C3 (#204885), factor I (FI) (#341280), C1q (#204876), goat anti-FB (#341272) and goat anti-FH (#341276) antiserum were obtained from Merck  and from healthy individuals (n=117) after informed consent in accordance with the Declaration of Helsinki. The study was approved by the National Ethical Committee (33986-1/2018 EKU). Characteristics of the patients and healthy donors are shown in 2SO4, absorbance was measured at 450 nm and at 620 nm as reference wavelength. To detect anti-MBL autoantibodies, serum samples were diluted in DPBS containing 10 mM EDTA. In the case of detecting anti-C1q autoantibodies, serum samples were diluted in DPBS containing 1 M NaCl to avoid interaction of C1q globular heads with IgG Fc parts. Samples were accepted as positive if the mean OD value for the investigated protein was at least two-fold greater than the mean OD value for the negative control protein alpha-1 antitrypsin. The isotypes of the autoantibodies were determined using monoclonal antibodies specific for IgG1, IgG2, IgG3, IgG4, IgGκ and IgGλ.Autoantibodies were detected using ELISA method, as described by Józsi and Uzonyi . Brieflyin vivo formed FB–FB-autoantibody complexes were removed. To this end, FB-specific antibodies isolated by Protein G column from goat anti-FB antiserum were immobilized on NHS column . The patient’s and control’s IgG fractions were loaded onto the anti-FB column, the flow-through fraction was collected and tested for the presence of FB.IgG fraction of the serum samples were isolated by Protein G column  according to the manufacturer’s instructions. For functional analyses, the To analyze FHR1- and/or FHR3-deficiency, serum samples at a dilution of 1:50 were separated on 10% SDS-PAGE under non-reducing conditions and transferred to nitrocellulose membrane (BioRad). After blocking with 1% BSA, 4% non-fat milk powder in DPBS-0.05% Tween-20, FHR3 and FHR1 were detected with rabbit anti-FHR3  and goat anti-FH antisera and the corresponding HRP-conjugated secondary antibodies, respectively. The blots were developed with the ECL detection kit (Merck).in vivo formed immune complexes, IgG fractions of the autoanti-FB positive patient and healthy controls were separated on 10% SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose membrane. After blocking with 1% BSA, 4% non-fat milk powder in DPBS-0.05% Tween-20, the membranes were incubated with goat anti-FB antiserum and HRP-conjugated rabbit anti-goat antibodies. These immune complexes were removed before further experiments to prevent their interference with the assays.To detect 2, 0.1% Tween-20) for 1 hour at 37°C. Convertase formation was detected with goat anti-FB and HRP-conjugated rabbit anti-goat antibodies. Convertase activity was determined by adding 10 µg/ml C3 for 1 hour at 37°C. The generated C3a was measured by the C3a EIA kit (Quidel). In some assays, patient’s or control IgG was added together with convertase components to analyze their effect on convertase formation. Convertase decay was determined after incubation of the formed convertase with the isolated IgG fractions alone or in the presence of 1 µg/ml FH for 1 hour at 37°C. Remaining convertase was detected with goat anti-FB antiserum.The solid phase AP C3 convertase was built up as described by Hourcade et al. in 2002  with mod6 rabbit red blood cells  as positive control for 15 min at 37°C. The serum was then added to the LPS-coated wells for 1 h at 37°C to determine the remaining complement activity. Complement deposition was measured with HRP-conjugated goat anti-C3 or mouse anti-C5b-9 and HRP-conjugated goat anti-mouse IgG.LPS  was immobilized at 10 µg/ml on Maxisorp microplate wells. Wells were washed with DPBS and blocked with 2% BSA. 10% complement active normal human serum (Quidel) was preincubated with the FB-free autoantibodies or with 5×106 RRBC were incubated with 7% complement active normal human serum (Quidel) in HEPES buffer  with serial dilutions of purified IgG for 30 min at 37°C. After centrifugation, optical density of the supernatants was measured at 414 nm.5×10p value < 0.05 was considered statistically significant. Comparison of gender distribution between RA patients and healthy controls were determined by χ2 test. To compare continuous variables (age), Mann–Whitney U test was performed. Comparison of FHR1- and/or FHR3-deficiency distribution between RA patients and healthy donors were performed by χ2 test. For comparison of the effects of IgG isolated from RA547 or healthy controls, one-way ANOVA was used with Dunnett’s multiple comparison test.Statistical analyses were performed with GraphPad Prism version 6.01 for Windows . A To investigate the presence and role of complement-specific autoantibodies in RA, we screened a Hungarian RA cohort for autoantibodies against FH, as well as C1q and MBL which were described as autoantibody targets in RA –35. In aHealthy control samples were negative for all tested antibodies, and we did not detect autoantibodies against FH, FI, C3b or the C3bBbP convertase in this RA cohort Table 3.in vivo, the isolated IgG fraction was analyzed by Western blot. A strong band corresponding to FB was detected in the RA547 sample, indicating the formation of FB–anti-FB immune complexes in vivo. , which are sensitive to lysis in normal human serum, were used. RRBCs were mixed with normal human serum in a buffer containing Mgt manner Figure 5via immune complexes present in the IgG preparations. However, compared to the IgG of healthy individuals, the autoanti-FB-containing patient IgG further decreased both C3- and C5b-9-deposition, presumably via inhibitory effects of the anti-FB autoantibodies . The patients/participants provided their written informed consent to participate in this study.ATM and BU performed experiments, analyzed the data, prepared the figures. AF, BR, GN took care of the patients and provided serum samples. GS, MJ and BU initiated the study, supervised the research. ATM, BU and MJ wrote the manuscript with help of the other authors. All authors read and approved the manuscript. All authors contributed to the article and approved the submitted version.


Flavivirus genus, has re-emerged as a global public health concern. The envelope (E) proteins of flaviviruses play a dual role in viral assembly and entry. To identify the key residues of E in virus entry, we generated a ZIKV trans-complemented particle (ZIKVTCP) system, in which a subgenomic reporter replicon was packaged by trans-complementation with expression of CprME. We performed mutagenesis studies of the loop regions that protrude from the surface of the virion in the E ectodomains . Most mutated ZIKVTCPs exhibited deficient egress. Mutations in DII and in the hinge region of DI and DIII affected prM expression. With a bioorthogonal system, photocrosslinking experiments identified crosslinked intracellular E trimers and demonstrated that egress-deficient mutants in DIII impaired E trimerization. Of these mutants, an E-trimerization-dead mutation D389A that nears the E-E interface between two neighbouring spikes in the immature virion completely abolished viral egress. Several mutations abolished ZIKVTCPs’ entry, without severely affecting viral egress. Further virus binding experiments demonstrated a deficiency of the mutated ZIKVTCPs in virus attachment. Strikingly, synthesized peptide containing residues of two mutants (268-273aa in DII) could bind to host cells and significantly compete for viral attachment and interfere with viral infection, suggesting an important role of these resides in virus entry. Our findings uncovered the requirement for DIII mediated-E trimerization in viral egress, and discovered a key residue group in DII that participates in virus entry.Zika virus (ZIKV), a family member in the  Flavivirus genus, which also include members such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV) [Zika virus (ZIKV), a re-emerging human pathogen, belongs to the us (WNV) . ZIKV waus (WNV) ,3. ZIKV us (WNV) . There iZIKV is a single-stranded, positive-sense RNA virus. Its genome is translated into a polypeptide that is processed by virus and host proteases to produce structural proteins  and non-structural proteins  ,7. The cZIKV E protein is the main membrane protein on the virus surface. It is composed of an N-terminal ectodomain and the C-terminal transmembrane helix (TM). The ectodomain includes the central domain (DI), a dimerization domain (DII), and an immunoglobulin-like domain (DIII). Flaviviruses invade cells through E-mediated receptor binding ,14. AlthMany residues of E have been reported to affect entry . Glycosytrans-complemented particle (ZIKVTCP) system, in which a ZIKV subgenomic replicon RNA bearing a secreted Gaussian luciferase (sGluc) reporter was packaged by CprM-E complementation. By using this ZIKVTCP system, we performed mutagenesis of the E loop regions that protrude from the surface of the virion and may serve as putative receptor-binding sites. We found that most mutations caused defects in viral egress, including mutations in DII and in the hinge region of DI and DIII that affected prM expression and mutations in DIII that affected E trimerization. We found mutations that impaired virus attachment without severely affecting virus egress. Further virus binding and peptide competing experiments demonstrate that residues 268–273 in DII contribute to virus attachment.Given the dual requirements for E protein in virion assembly and virus entry, it is difficult to dissect the residues of E solely involved in virus entry. To identify the key resides that participate in virus entry, we first generated a ZIKV HA and phCMV-prM-EHA, respectively. Three-Alanine (AAA) mutations were introduced into E in the plasmid phCMV-CprM-EHA by fusion PCR-mediated mutagenesis to get the plasmids phCMV-CprM-EHA-Mutants. The suppressor tRNA plasmid (pSVB.Yam) and the amino-acyl tRNA synthetase plasmid for p-azido-L-phenylalanine (pcDNA.RS) were kindly gifted by Thomas P. Sakmar (Rockefeller University) and have been described previously [HA by fusion PCR-mediated mutagenesis. The L107TAG mutation was introduced into plasmids phCMV-CprM-EHA-mutants (Mut8 to Mut43) to get the plasmids phCMV-CprM-EHA-L107TAG-mutants (Mut8 to Mut43), respectively. The infectious clones of ZIKV C7 and C7-Gluc plasmid has been described previously [HA and C7.EFlag, respectively. The details of the mutation were described in Supplementary Table 1.To generate the plasmids phCMV-CprM-E, phCMV-prM-E, the coding regions of CprM-E and prM-E were amplified from a ZIKV infectious clone  and theneviously . The TAGeviously . The codin vitro-transcribed viral RNAs into Vero cells. The C7.EHA and C7.EFlag were generated by transfection of in vitro-transcribed viral RNAs into Vero cells with transfection reagent . The virus titres were determined in Vero cells by plaque assay as described [ZIKV C7, C7-Gluc were generated by electroporation of escribed .TCPs, sgZIKV-sGluc-Vero cells (0.75 × 105) in 48-well plate were transfected with 0.26 μg phCMV-CprM-EHA (WT), phCMV-prM-EHA (ΔC) or phCMV-CprM-EHA-mutant plasmids by TransIT®-LT1 Transfection Reagent . Four days later, the supernatant was collected and centrifuged at 500 g for 10 min to remove the debris and stored at −20°C.To generate ZIKV6 sgZIKV-sGluc-Vero cells in 10-cm dish were transfected with 15 μg phCMV-CprM-EHA or the phCMV-CprM-EHA mutants plasmids using the TransIT®-LT1 Transfection Reagent , 4 days later, the supernatants were collected into a 15 mL centrifuge tube and centrifuge at 1000× g for 10 min to remove debris. About 11.2 ml clarified supernatants were loaded on the top of 2 mL 20% sucrose cushion (W/V in PBS). Centrifugation was performed with SW41 rotor  at 247,600× g for 4 h at 4°C with no brake. After gently removal of the supernatants, ZIKVTCP pellet was resuspended in 300 μl PBS, aliquot, and stored at −80°C.About 3.6 × 10HA  or the C7.EHA-normalized ZIKVTCPs at 4°C for 2 h. After being washed six times with PBS, the cells were either harvested for quantification of viral RNA or fixed for immunostaining.Vero cells were infected with C7.EALEAEM DGAKG RLFSG HLKCR LKMDK LRL) and P1-Mut (VLGSQ EGAVH TALAG AAAAAA DGAKG RLFSG HLKCR LKMDK LRL) were synthesized by GL Biochem (Shanghai) Ltd and dissolved in PBS to make a 0.5 mg/ml stock. Vero cells (0.75 × 105) were seeded onto 48wps and incubated at 37°C overnight. The cells were washed with PBS and incubated with 10 μM P1 or P1-Mut for 2h at 4°C, then infected with C7-Gluc  or C7.EHA-normalized ZIKVTCP for 2h at 4°C. The supernatants were discarded, and the cells were added with fresh media. The luciferase activity was measured 4 days later.For peptide competing for viral infection, peptides P1  or C7  for 2h at 4°C. After been washed with PBS for six times by centrifugation at 1100g for 5 min, RNAs were extracted by TRIzol reagent and subjected to reverse transcription and Real-time PCR assay.For peptide competing for virus binding, Vero cells are digested by trypsin, resuspended in PBS with 1% FBS and adjusted to a cell concentration of 1.2 × 10ALEAEM DGAKG RLFSG HLKCR LKMDK LRL), FITC-P1-Mut: (FITC-VLGSQ EGAVH TALAG AAAAAA DGAKG RLFSG HLKCR LKMDK LRL), FITC-P2: (FITC-GRLIT ANPVI TESTE NSKMM LELDP PFG) and FITC-P2-Mut (FITC-GRLIT ANPVI TESTE NSAAA LELDP PFG) were synthesized by GL Biochem (Shanghai) Ltd and dissolved in PBS to make a 0.5 mg/ml stock. Various concentrations of peptides were incubated with Vero cells for 2h at 4°C. Then, the peptides were removed, and the cells were washed with PBS four times and then fixed with 4% paraformaldehyde for 15 min and observed by fluorescence microscopy.Peptides FITC-P1  and anti-CD44 (1:300) antibodies at 4°C overnight. After being washed three times with PBS containing 3%BSA, samples were incubated with Alexa 488- and Cy3-conjugated secondary antibody at 37°C for 1 h. After being washed thrice with PBS, samples were incubated with 2 ml PBS containing 2 μl Hoechst 33342  for 3 min to stain the nucleus. After being mounted on the coverslips, the cells were observed using a confocal laser scanning microscope . Images were acquired and analyzed by using Image J software as previously described .5 Vero cells on 6-cm dish were cotransfected with 4.3 μg CprM-E expressing plasmids, 4.3 μg pSVB.Yam and 1.2 μg pcDNA.RS by TransIT-LT1 (Mirus) at a ratio of 1:3 (plasmid: reagent). Nine hours after transfection, the medium was changed to DMEM containing 10% FBS and 0.5 mM azF. Forty-eight hours post transfection. The cells were washed with PBS and then irradiated on ice under 365 nm UV light by a UV lamp  in a dark room for 30 min. The UV light energy given out by the lamp is 3.405 Ev and the sample is 20 mm away from the UV lamp. Then, the cells were lysed with 300 µl IP buffer . After disruption by passing through a 27-gauge needle 20 times and clarification by centrifugation at 15,000g for 10 min at 4°C, 300 μl of the soluble fraction was incubated overnight at 4°C with 10 µl anti-HA magnetic beads . Beads were washed 4 times in 500 µl wash buffer , and then lysed in 2 × SDS loading buffer. The samples were boiled for 10 min and then analyzed by Western blotting.Photo crosslinking was carried out as previously described . BrieflyStatistical analysis was performed with the GraphPad Prism 8 software. Specific tests are described in the figure legends.trans-complemented particle system, in which the viral structural proteins CprM-E are expressed in ZIKV subgenomic replicon cells and the viral subgenomic replicon RNA is then packaged by the CprM-E trans-complementation. Trans-complemented particle system is a widely used strategy to dissect the assembly and entry process in flavivirus, especially when some E mutations abolished virus rescue in the reverse genetic systems and thus it is impossible to generate an E-mutated virus [Trans-complemented particle system is a widely used molecular biology tool to dissect the assembly and entry process in flavivirus, although it doesn’t quite represent a real virus [Gaussia luciferase (sGluc), FMDV 2A peptide (2A) and blasticidin resistant gene (BSD). The coding sequence of the first 25 residues of capsid was retained. The EMCV IRES was inserted before the open reading frame of the polypeptide of viral non-structural proteins NS1-NS5. The C-terminal 24 resides of E was retained to function as the signal peptide (NS1 sig) of NS1. The self-cleaving RNA sequence  was used to generate the genuine 3’-terminal nucleotide of the viral genome after in vitro transcription. To assess the replication of the subgenomic RNA, the in-vitro-transcribed replicon RNA (sgZIKV-sGluc) and a mutant bearing the RNA-dependent-RNA polymerase inactive mutations (sgZIKV-sGluc-GNN) were transfected into Vero cells. Luciferase activities in the supernatants were measured at various time points post transfection. Comparing with sgZIKV-sGluc-GNN RNA, the sgZIKV-sGluc RNA exhibited robust replication, whereas the sgZIKV-sGluc-GNN only supported initial translation of viral RNA  in the supernatants were used to infect naïve Vero cells ). The C7-EHA and C7-EFlag virus replicated similarly to C7 virus in Vero cells, as evidenced by comparable viral protein levels in the infected cells ). The expression of HA and Flag tags was verified by Western blotting ). The C7-EHA and C7-EFlag virus also plaqued similarly to C7 in Vero cells ). In the following studies, we chose the EHA and generated ZIKVTCP using CprM-EHA (ZIKVTCPHA) as the method described above. Viral replication in the ZIKVTCP- and the ZIKVTCPHA-infected cells was examined by Western blotting analysis of the cell lysate and measuring the luciferase activity in the supernatants of the infected cells. There were comparable NS5 and E levels in the infected cells and comparable luciferase activity in the supernatants  and TCPHA enters similarly to ZIKVTCP. The entry of ZIKVTCPHA was also sensitive to bafilomycin A1 and NHCl4 ). To further confirm that the insertion of the HA tag does not affect the virus assembly and entry, we transfected the same amount of prME or prMEHA into sgZIKV-sGluc-Vero cells, and the ZIKVTCPHA exhibited similar egress and cellular binding as the ZIKVTCP ). Taken together, we generated a ZIKV trans-complemented particle system with HA-tagged E protein.To facilitate detection of E, we inserted an HA tag or Flag tag into a tolerant site after the residue 147 of E as previous reported . The insTCPHA system, we focused on the loop regions of the DIII that had been proposed to be involved in virus attachment and entry [HA mutants or prM-E (ΔC) into the Vero-sgZIKV-sGluc cells as described above. The egress of the ZIKVTCPsHA was assessed by measuring the intracellular expression of the E mutants and the released E in the supernatants after anti-HA immuno-capture. The infectivity of the released ZIKVTCPsHA was determined by measuring the sGluc activity in the ZIKVTCPHA-infected cells . The observation that most E mutants impaired ZIKVTCPs egress is consistent with the previously proposed, participation of E in virion assembly [Consistent to a previous study, the residues within the Mut 1 and Mut 2 are tolerant to exogenous insertion , Mut 1 aassembly .Flavivirus Capsid (C)-prM is processed by viral NS2B-3 protease in the cytosol and host signalase in the ER lumen. The signalase cleavage at C-prM is delayed until the viral protease cleaves the upstream of the prM signal sequence, which results in the production of prM and the capsid anchor (Ca) ). Ca/pr cleavage is regulated by E expression and mutations in prM impair virion assembly [TCP egress, we examined the impact of these E mutants on prM-E processing. We transfected the CprM-EHA mutants and prM-EHA (ΔC) into Vero-sgZIKV-sGluc cells and examined the prM-EHA processing by Western blotting analysis of EHA and prM. Most E mutants were processed similarly as wild type (WT) E, except for Mut 42  and Supplementary Figure 4(a–c)). Mut 42 generated a band with smaller molecular weight than expected ) (See below). Strikingly, Mut 4, Mut 7, Mut 8, Mut 11, Mut 13, Mut 14, Mut 15, Mut 17 Mut 18 and Mut 19 severely caused the reduction of prM expression  and 4).assembly ,49–52. AAt neutral pH in the ER lumen, the immature virion particle is composed of trimeric prM-E heterodimer. At acid pH in the trans-Golgi network (TGN), prM-E trimer undergoes conformation change into E homodimer, exposing the pr/M cleavage site and resulting in the furin-mediated pr-M cleavage and virion maturation ,11,53.298V299S300Y301S302) in the assembly-dead mutants Mut15, Mut16, and residues (G333P334) in the Mut26 and Mut27 reside in the E-E interface within the prM-E trimer  was restored  were observed. Obvious trimeric E crosslinking occurred via residues within the E dimer interface or the E trimers, including W101, G102, C105, G106, L107, G224, A225, D226, T227, G228, T229, G259, S260, K297, G298, V229, S300 and Y301 ). The reduced crosslinking by these residues is probably either due to the weak crosslink per se or due to the replacement of these residues by AZF that impairs E dimerization or trimerization. Notably, there were specific irradiation-induced crosslinked bands with molecular weight equivalent to that of dimeric prME (148 KD) and prME (74 KD)  ), which is likely due to the lack of Capsid-prM processing by NS3 and aberrant virion particle assembly.We also monitored crosslinking via the residues CHA mutants  were analyzed. The D389A mutation nearly completely abolished ZIKVTCPs egress. Mut 42, G388A and K390A significantly reduced ZIKVTCPs egress (see discussion). These results suggest a critical role of D389 in E trimerization and egress.We further focused on the Mut 42 and did mutation on each residue to assess the effect on E trimerization ). We used the normalized ZIKVTCPs to infect Vero, Huh7 and A549 cells. Viral infection was determined by measuring luciferase activity in the infected cells. Compared with ZIKVTCP-WT, the ZIKVTCP-mutants barely infected these cells, except that ZIKVTCP-Mut36 replicated to a limited level in Vero cells  that contains the mutated residues in Mut 9 and Mut 10 and FITC-P2-Mut that contains the mutated residues in Mut 37, respectively  with lower fluorescence intensity and the group 2 (G2) with higher fluorescence intensity. The G2 populations raised with the increase of peptide concentration, with significantly higher populations in the FITC-P1-bonund cells than that in the FITC-P1-Mut-bonund cells ). As the P1 exhibited higher binding affinity to Vero cells, then we used P1 peptide to compete virus for cellular attachment and viral infection. We incubated Vero cells with various concentrations of P1 and P1-Mut peptides, and then infected the cells with wild-type Zika virus C7 and ZIKV reporter virus C7-Gluc at 4°C for 2 h. The cellular attachment was evaluated by quantifying the bound viral RNAs  in the mature virion and residues near the prM-E interface within the trimeric spike (C105G106L107) in the immature virion to mediate photocrosslinking of E trimers and putative prME dimers (105G106L107) near the prM-E interface within the trimeric spike in immature virions are probably a mixture of mature E-dimer and immature E-trimer, as these residues are also near the E dimer interface in the mature virion  that contains the residues corresponding to the Mut 9, Mut 10 and peptide (P2) that contains residues corresponding to the Mut 37 bound to Vero cells, whilst P2 exhibited less binding than P1 (TCP at a concentration of 10 μM (268L269E270A271E272M273 (Mut9 and Mut10) play an important role in virus entry at the attachment step, although we can’t exclude their also having roles at a post-attachment step. Given the Mut37 dramatically reduced attachment, as determined by quantification of the bound viral RNA  system, we performed mutagenesis of the loop regions in the envelope (E) protein ectodomains, for their roles in viral egress and entry. We identified key resides that affect viral egress and entry. We unveiled the requirement for DIII in E trimerization and virus assembly and uncovered resides (268-273aa in DII) that play important role in virus attachment. Our findings advance understanding of the mechanism of flavivirus entry and egress.In summary, in a ZIKV Click here for additional data file.


During an investigation in October 2018, two people with diarrhoea, mild abdominal pain, and mild arthralgia symptoms in Guangxi, China, were identified as infected by H9N2 avian influenza virus (AIV). Four H9N2 AIVs were isolated from one of two patients, a pet cat, and a dead chicken (two respective isolates from its lung and kidney tissues) bred by the patients at a backyard farm. Epidemiological investigation indicated that the newly bought chicken died first, and clinical syndromes appeared subsequently in the two owners and one cat. Furthermore, the two individuals possessed high H9N2-specific hemagglutination inhibition and microneutralization antibodies. Shared nucleotide sequence identity (99.9% – 100%) for all genes was detected in the four H9N2 isolates, and hemagglutinin (HA) T138A located on the receptor binding domain (RBD), resulted from nucleotide polymorphisms that were exclusively found in the isolate from the female patient. Moreover, HA K137N on the RBD was found in isolates from these three host species. Importantly, these four H9N2 isolates presented an exclusive binding preference for the human-type receptor (α2-6-SA), and could replicate and cause pathological changes in mice. Phylogenetic analyses showed that these four isolates clustered together and belonged to clade C1.2, lineage Y280. In addition, H9N2 viruses of human origin are genetically divergent and interspersed with the widespread poultry-origin H9N2 AIVs. All these results indicate a high risk of H9N2 AIVs in public health, and effective prevention and control measures against H9N2 AIVs should be considered and performed for both animal and human health. The A/H9N2 (hereafter referred to as H9N2) low-pathogenicity avian influenza virus (LPAIV) is of great global concern since its worldwide circulation and even long-term endemicity in the poultry populations of countries in Asia, Africa, and the Middle East . Based ohttps://www.who.int/teams/global-influenza-programme/avian-influenza/monthly-risk-assessment-summary) [https://www.who.int/teams/global-influenza-programme/avian-influenza/monthly-risk-assessment-summary) [Occasional cross-species transmission of H9N2 AIVs from poultry to mammals, especially humans, has been reported ,9–11. Sisummary) . Typicalsummary) ,16. Furtsummary) . The novsummary) . The facIn the present study, we report a cluster of H9N2 infections among two humans, one pet cat, and several chickens at a backyard farm in Guangxi Province in October 2018. An epidemiological investigation and hemagglutination inhibition (HI) and microneutralization (MN) assays were performed to determine the disease progression of H9N2 human infections on the farm. Molecular characteristics analyses and the receptor-binding and virulence-related assays were also employed to understand the infective features and to explore the potential transmission routes of the H9N2 virus among chickens, humans, and cats. The findings of our study further uncover the public health risks of H9N2 and support the development of targeted prevention and control strategies.Animal studies were conducted according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology, China. In this study, the animal experiments were approved by the Research Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences (APIMCAS2018014).Ten-day-old specific pathogen-free (SPF) embryonated chicken eggs were obtained from Beijing Vital River Laboratory Animal Technology Company (China) and used for virus isolation at 37 °C and 80% humidity.Lung and kidney samples of the dead chicken were collected on October 15, 2018. Throat swabs from the two patients were collected on October 19. On the same day, the diseased cat was euthanized, and heart, liver, spleen, kidney, lung, and larynx samples were collected. The supernatants of the swabs and tissue homogenates were inoculated into 10-day-old specific pathogen-free chicken embryos through the allantoic cavity inoculation route, as previously described [Viral RNA was extracted from the allantoic fluid of the AIV-positive samples, including throat samples from the female patient, heart samples from the cat, and lung and kidney samples of the dead chicken, using the MagaBio plus Virus RNA Purification Kit . The whole genome of the AIV isolates was sequenced using next-generation sequencing (NGS). Specifically, viral RNAs were used for RT-PCR, and DNA synthesis was conducted as previously described ,2. SequeSequencing reads generated by NGS were processed and assembled to obtain sequence information on H9N2 AIVs. First, the low-quality reads , adaptor-contaminated reads (> 5 bp matched to the adaptor sequence in either of the paired reads), poly-Ns (with 8Ns), and duplicated reads were discarded from the raw read data using SOAP2 v2.21 . The clehttps://nmdc.cn; accession number: NMDCN0001181 – NMDCN0001190) and the EpiFlu database of GISAID .The four H9N2 isolates were named A/Guangxi/NN10.19T-NGS/2018, A/cat/Guangxi/NN10.19H-NGS/2018, A/chicken/Guangxi/NN10.19L1-NGS/2018, and A/chicken/Guangxi/NN10.19K2-NGS/2018. The corresponding gene sequences obtained in this study are deposited in the National Microbiology Data Center  and α2,6 sialic acid receptor  as described previously [To further confirm virus receptor-binding specificity, we performed hemagglutination assays according to the manual from the World Health Organization (WHO) using 1% chicken red blood cells , 1% resialylated cRBCs (α2-6-SA receptors), and 1% sheep red blood cells  ,28. The Plasma samples collected from the two patients were treated with the receptor-destroying enzyme (RDE). We tested the plasma samples for antibodies against the H9N2 isolate (A/Guangxi/NN10.19T-NGS/2018), and representatives of influenza A virus, namely, A/H1N1 (A/Brisbane/02/2018), A/H3N2 (A/Kansas/14/2017), and A/H5N6 (A/Shenzhen/TH002/2015), as well as influenza B viruses, B/Colorado/06/2017 (belonging to B/Victoria/2/87 lineage labelled as Bv hereafter) and B/Phuket/3073/2013 (belonging to B/Yamagata/16/88 lineage labelled as By hereafter), using HI and MN assays, respectively, according to the manual provided by the WHO  and a pr6 EID50 of H9N2 viruses isolated from the chicken (A/chicken/Guangxi/NN10.19L1-NGS/2018), cat (A/cat/Guangxi/NN10.19H-NGS/2018), and human (A/Guangxi/NN10.19T-NGS/2018), respectively. One control group of mice was intranasally inoculated with PBS. Three mice in each group were euthanized on 3, 5, and 7 days post-infection (dpi). The lungs of the euthanized mice were harvested and homogenized to detect viral titres  using qRT-PCR. The lung index (ratio of lung weight to body weight) was also calculated for each euthanized mouse. In addition, five mice in each group were monitored for bodyweight changes until 14 dpi. The percent bodyweight change was calculated as the ratio of bodyweight to initial bodyweight on day 0.Four groups of fourteen 7- to 8-week-old female BALB/c mice  were used to examine the pathogenicity of H9N2 viruses identified in this study. Three groups of BALB/c mice were intranasally inoculated with 10For histopathological examinations, the left lungs collected from euthanized mice at 5 dpi were immersed in 4% paraformaldehyde. The fixed lung tissues were dehydrated, embedded in paraffin, cut into 4-μm-thick sections, and stained with haematoxylin and eosin (H&E) staining.To uncover the genetic divergence of H9N2 viruses isolated from the chicken, cat, and human at the backyard farm, we first calculated the percent identity between each H9N2 virus based on consensus nucleotide sequences. Next, we analysed the single nucleotide polymorphisms (SNPs) and amino acid substitutions resulting from the nucleotide polymorphisms in H9N2 virus populations isolated from the different hosts. We also analysed the key amino acid sites in the HA, NA, PB2, PA, and M genes to understand the potential biological features of H9N2 viruses, including the receptor-binding preference, replication, virulence, and antiviral drug susceptibility. Subsequently, we inferred the structure of the ectodomain of HA molecule of A/Guangxi/NN10.19T-NGS/2018, using the homology modelling method with A/swine/Hong Kong/9/98  as a template in the SWISS-model . The thrhttps://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi#mainform) and the EpiFlu database of the GISAID as of November 22, 2020. Additionally, we downloaded the nucleotide sequences of H9N2 AIVs isolated from humans in China. Based on previous studies, we also gathered the representative strains for H9N2 lineage classification: the BJ/94 lineage (represented by A/chicken/Beijing/1/1994), Y280 lineage (A/duck/Hong Kong/Y280/1997), G1 lineage (A/quail/Hong Kong/G1/1997), and Y439 lineage (A/duck/Hong Kong/Y439/1997) [The nucleotide sequences of eight gene segments of H9N2 AIVs isolated in mainland China were downloaded from the influenza virus database hosted by the National Center for Biotechnology Information NCBI; 39/1997) . We remo39/1997) . We cons39/1997) . Limited39/1997) . To unde997 . We During our surveillance on AIVs, two farmers presented some syndromes who bred chickens in their backyard, so we collected samples from them and their cats and chickens for the identification of pathogens. And we collected demographic characteristics  of the two humans living on the farm, and the geographic location. We also recorded the clinical symptoms, onset dates, course of disease development in the humans, cat, and chickens and their exposure and contact history.On October 6, 2018, seventy 120-day-old chicks were imported into a backyard farm that already had two chickens . Three dThe H9N2 AIVs were isolated from the throat swab of the female patient, heart sample from the pet cat, and lung and kidney samples from the dead chicken after 1–3 consecutive passages.The levels of HI and MN antibodies against the H9N2 and other influenza viruses  in the plasma samples of the two recovered humans were detected . The plaTo understand the potential mechanism behind the interspecies transmission of H9N2 AIVs on the same farm, we performed solid-phase receptor-binding detection and hemagglutination assay using the red blood cells with different types of sialic acid receptors to test the receptor-binding properties of the H9N2 isolates. In the solid-phase receptor-binding assay, the H9N2 viruses isolated from the human, cat, and chicken showed an exclusive binding preference for the human-type receptor (α2-6-SA) rather than the avian-type receptor (α2-3-SA) . The samp-value < 0.05 given Student’s T-test) higher than those of mice infected with PBS in the control group at 7 dpi. H&E staining revealed the pathological changes in the lungs  of mice challenged with the H9N2 viruses.Mice infected with H9N2 viruses isolated from the cat and human showed non-negligible body weight losses from 3 dpi to 9 dpi compared to mice inoculated with PBS or H9N2 virus from the chicken . The lunConsidering the consensus nucleotide sequences, the percent sequence identities between each pair of H9N2 AIVs isolated in this study were over 99.9% for each of the eight genes: HA (99.94% – 100%), NA (99.93% – 100%), PB2 (99.96% – 100%), PB1 (100%), PA (99.95% – 100%), MP (99.90% – 100%), NP (100%), and NS (100%).To further uncover the genetic diversity of the virus populations, we found ten nucleotide polymorphisms specific to H9N2 isolated from the human and cat in comparison with those from the chicken given the NGS data . Three oFurthermore, all four H9N2 AIVs isolated in this study possessed 226L, 155 T, and 183N in the HA gene sequences  circulating in poultry farms and live poultry markets. Surveillance of human H9N2 infections, especially in workers occupationally exposed to poultry, should be enhanced to monitor the emergence of human-adapted H9N2 viruses and provide early warning of a potential outbreak or pandemic.Click here for additional data file.


In the original publication, Figure 8D/H/I/J and Figure 10B/C/D/E showed the MDA, AST, ALT, and LDH levels in APAP-induced acute hepatitis model and RFP-induced chronic hepatitis model, respectively. We are sorry that Figure 10B/C/D/E were presented the same as Figure 8D/H/I/J by mistake. The corrected Figure 10B/C/D/E are shown below. The authors confirm that the correction would not change the result and conclusion of this article. The authors sincerely apologize for any inconvenience or misunderstanding that may have caused.


To investigate the changes of human leukocyte antigen DR (HLA-DR) and CD38 coexpression subsets on T lymphocytes following interferon (IFN) therapy for those who have chronic hepatitis B (CHB).A prospective cohort of CHB patients participated in this study. CHB patients without IFN treatment  were given pegylated interferon alfa (Peg-IFNα) treatment. Peripheral blood samples were taken at baseline, 4 weeks and 12-24 weeks of Peg-IFNα treatment. For the patients who entered the Peg-IFNα plateau phase due to the stagnation of the decrease in HBsAg, and Peg-IFNα was discontinued and Peg-IFNα therapy was resumed after an interval of 12-24 weeks. During the interval, they received first-line NAs treatment. Peripheral blood samples were collected at the baseline of the plateau phase, 12-24 weeks of intermittent treatment, and 12-24 weeks of Peg-IFNα retreatment. The peripheral blood samples were taken to determine virological, serological and biochemical indices of hepatitis B virus (HBV), and T lymphocyte related phenotypes were detected using flow cytometry.+CD38dim subsets increased significantly at first, then decreased gradually, while the percentage of HLA-DR+CD38hi subsets markedly increased. During long-term Peg-IFNα treatment, there was a considerable negative correlation between HBsAg and the HLA-DR+CD38hi subset percentage. The persistent high proportion of HLA-DR+CD38hi subsets was related to the occurrence of Peg-IFNα plateau phase. After Peg-IFNα intermittent treatment, the percentage of HLA-DR+CD38hi subsets decreased significantly. After Peg-IFNα retreatment, the level of HBsAg began to decrease again. At the same time, the percentage of HLA-DR+CD38hi subsets significantly increased, but it was still lower than that at the baseline level.In the process of long-term treatment of Peg-IFNα, the percentage of HLA-DR+CD38hi subsets on T lymphocytes. IFN intermittent treatment could significantly reduce the proportion of HLA-DR+CD38hi subsets, helping regain the antiviral efficacy of IFN during IFN retreatment.The spectrum of HLA-DR and CD38 coexpression subsets on T lymphocytes changed during the long-term treatment of IFN. The establishment of the IFN plateau phase was linked to the persistence of a considerable proportion of HLA-DR Millions of people, especially those in China, are infected with the chronic hepatitis B virus (HBV). Around 86 million people in China have chronic HBV infection, including 30 million who have chronic hepatitis B (CHB), and 400 thousand people die from HBV-related causes per year , 2. In aHowever, although many studies have indicated that long-term IFN antiviral therapy increases hepatitis B surface antigen (HBsAg) clearance, which helps to achieve long-lasting immune control and clinical cure , 11. The+ T lymphocytes declined dramatically while receiving long-term IFN therapy, and they primarily lost subsets that were in the stage of terminal differentiation  lymphocytes, dendritic lymphocytes, and T lymphocytes all participate in the immune system’s ability to eliminate HBV, with T lymphocyte activation playing the most significant role in virus clearance –15. Immantiation . HoweverThis study’s major goal is to investigate how HLA-DR and CD38 coexpression subsets proportion on T lymphocytes fluctuates dynamically throughout IFN-based treatment of CHB. The findings of this study will aid in explaining why IFN antiviral therapy’s efficiency varies at different phases.CHB Patients hospitalized at Beijing Ditan Hospital’s Second Department of Hepatology between June 2021 and August 2022 made up the prospective cohort. According on their course of therapy, the patients were categorized into three groups: The Naïve group consisted of CHB patients who had never undergone antiviral medication, while patients who had only ever undergone first-line NAs antiviral medication made up the NA-treated group. Patients who had previously undergone pegylated interferon alfa (Peg-IFNα) therapy were enrolled in the Plateau group due to the failure of HBsAg to continuously decrease.In the Naïve group, patients began to receive subcutaneous injection of Peg-IFNα-2a 180 μg/week, and selected first-line NAs antiviral drugs  according to their clinical data and personal wishes for combined treatment. Peg-IFNα-2a 180 μg/week was administered subcutaneously to patients in NA-treated group on the basis of NAs antiviral drugs. Blood samples were taken from patients in Naïve group and NA-treated group at baseline, 4 week and 12-24 weeks after IFN treatment. The HBV virological and serological indices, biochemical indices, and phenotypes associated with T lymphocytes were identified. In the Plateau group, when HBsAg decreased to the plateau phase, the treatment of IFN was suspended and entered into intermittent treatment for 12-24 weeks, during which only ETV (0.5 mg/day) or TDF (300 mg/day) or TAF (25 mg/day) was used to maintain the antiviral efficacy, and after the interval, IFN was retreated on the basis of NAs. Blood samples were taken from patients in Plateau group at the baseline of plateau, during intermittent treatment of 12-24 weeks and during 12-24 weeks of IFN retreatment. The HBV virological and serological indices, biochemical indices, and phenotypes associated with T lymphocytes were identified. In addition, liver ultrasonography was performed every 12-24 weeks at baseline and during the follow-up period.In addition, the comparison between the Plateau group and its corresponding control group  could more accurately portray the advantages of IFN intermittent therapy and the relationship between the changes of T lymphocyte-related surface markers and clinical efficacy during this period. However, patients who HBsAg levels decreased < 1 lg IU/mL after 24 weeks of Peg-IFNα therapy should stop using IFN . Even ifThe Ethics Committee of Beijing Ditan Hospital, which is a part of Capital Medical University, granted approval for this study (Jing Di Lun Ke Zi 2018 NO. 023-01), and it was registered as a clinical trial (NCT04028856). It was designed and implemented according to Helsinki standards. All selected patients have signed informed consent.Inclusion criteria (1): 18-65 years old; (2) HBsAg positive lasting more than 6 months (≥ 0.05 IU/mL). In addition, it was also necessary for Naïve group: (1) never received antiviral therapy; it was also necessary for NA-treated group: (1) so far and only take first-line oral antiviral therapy more than 6 months. For Plateau group, it also needed to be satisfied: (1) the level of HBsAg decreased to the plateau period after IFN antiviral treatment for more than 6 months. In order to be considered in a plateau period, the level of HBsAg must have dropped by 0.5 lg IU/mL from the preceding detection time point (more than 3 months) .Exclusion criteria: (1) previously known viral hepatitis (except hepatitis B) and epstein-barr virus, cytomegalovirus, human immunodeficiency virus and other non-hepatitis virus infections; (2) complicated with other liver diseases, for instance metabolic-related fatty liver disease, alcoholic hepatitis, autoimmune hepatitis, hepatolenticular degeneration, decompensated cirrhosis and liver cancer; (3) mental illness; (4) severe heart, brain, lung, kidney or other system diseases; (5) usage of other liver-damaging drugs and/or chronic alcoholism; (6) usage of hormones and/or immunosuppressants.The peripheral venous blood of the patient was collected at the specified time point and put into the EDTA anticoagulation tube. Using lymphocyte separation solution, peripheral blood mononuclear lymphocytes (PBMC) were isolated by density gradient centrifugation. All samples were processed and analysed within 24 hours after sample collection.-APC (Clone HB-7) . FACSCanto flow cytometry  and FlowJo software  was used to gather and analyze the data, respectively.PBMCs and direct coupling antibody were incubated together in dark environment at 4°C for 20 minutes. Then 2 mL of 1 × PBS were added to wash lymphocytes. After rotating 1200 rpm for 5 minutes, the supernatant was discarded. Finally, 200 mL of 1 × PBS was added to make the pre-test sample. The monoclonal antibodies were anti-human CD4-APC-Fire750 (Clone SK3), CD8-BV510 (Clone SK1), HLA-DR-PE-Cy7 (Clone L243), and CD38Roche CobasTaqMan 96 , an automatic real-time fluorescence quantitative polymerase chain reaction (PCR) detector, was utilized to detect serum HBV DNA load. Serum HBsAg and hepatitis B e antigen (HBeAg) levels were measured by Abbott Architect i2000 kits , and liver function indexes were detected by Hitachi 7600 automatic biochemical analyzer .P value was adjusted by Bonferroni method. Patients were taken as random effects, groups and measurement time points were taken as fixed effects. The standard model was selected by likelihood ratio test. It did not violate the standard model hypothesis. The adjusted model took age and sex as covariables to exclude potential confounding factors. The Spearman correlation test was used to get the P values and correlation coefficient. All meaningful analyses were double-tailed, and P < 0.05 was statistically significant.SPSS 21.0  and GraphPad5  software were used for statistical analysis. The categorical variables were expressed by frequency and percentage, and the chi-square test was used to compare the differences between groups. The normality of continuous variables was evaluated by Shapiro-Wilk test. Continuous variables that were normally distributed were represented by mean ± standard deviation (SD), and continuous variables that were not normally distributed were represented by the median and inter-quartile range . According to the normality of the baseline data, One-way ANOVA test and Kruskal-Wallis test were respectively used to compare the multiple groups at baseline, and then Tukey’s multiple comparisons test and Dunn’s multiple comparisons test were utilized to conduct pairwise comparisons between the groups. to make pairwise comparisons between the groups. The linear mixed effect model (restricted estimation maximum likelihood) was used to analyze the changes of parameters at different time points in each group, and the multiplicity of This study comprised 150 patients with CHB in total, including 53 patients in Naïve group, 51 patients in NA-treated group and 36 patients in Plateau group. P < 0.0001; P = 0.0122). Although the HBsAg level in the NA-treated group was marginally less than that in the Naïve group, there was no markedly significant variation (P = 0.0680). HBV DNA load was considerably higher in the Naïve group compared to the NA-treated group and Plateau group, respectively , but there was no remarkable variation in HBV DNA level between NA-treated and Plateau group (P = 0.9019). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in NA-treated group were notably lower than those in Naïve group and Plateau group respectively , but there was no discernible variation in terms of ALT and AST levels between Naïve group and Plateau group , as shown in In comparison to both the Naïve group and the NA-treated group, HBsAg amount in the Plateau group was much lower . In the NA-treated group, more HLA-DR+CD38-CD4+ T lymphocytes were present than those in the Naïve group (P = 0.0289). HLA-DR+CD38dim CD4+ T lymphocyte percentage in Plateau group was considerably greater than that in Naïve and NA-treated group , but no discernible variation was observed between Naïve and NA-treated group (P > 0.9999). The proportion of HLA-DR+CD38hi CD4+ T lymphocytes in Plateau group was considerably greater than that in Naïve and NA-treated group , but there was no discernible variation in the percentage of HLA-DR+CD38hi CD4+ T lymphocytes between Naïve group and NA-treated group (P > 0.9999). HLA-DR+CD38dim CD8+ T lymphocyte percentage in Plateau group was noticeably less than that in Naïve group and NA-treated group, respectively , however there was also no discernible variation in HLA-DR+CD38dim CD8+ T lymphocyte level between Naïve group and NA-treated group (P > 0.9999). The difference trend of HLA-DR+CD38- CD8+ T lymphocytes and HLA-DR+CD38hi CD8+ T lymphocytes in different groups was similar to that of CD4+ T lymphocytes. HLA-DR+CD38- CD8+ T lymphocyte percentage in Plateau group was considerably lower than that in Naïve and NA-treated group . In NA-treated group, HLA-DR+CD38- CD8+ T lymphocyte percentage was noticeably more than that in Naïve group (P = 0.0002). In comparison to the Plateau group, the HLA-DR+CD38hi CD8+ T lymphocyte percentage was considerably less in Naïve group and NA-treated group . HLA-DR+CD38hi CD8+ T lymphocyte proportion was not markedly different between Naïve group and NA-treated group (P > 0.9999), as shown in + and CD8+ T lymphocytes after long-term IFN treatment.In order to determine the level of T lymphocyte activation under different treatment conditions, we analyzed the coexpression of HLA-DR and CD38, representing the key T lymphocyte activation indicator. We defined three subsets in activated HLA-DRsubgroup Figure 1+ and CD8+ T lymphocytes during IFN therapy, we followed up these patients who chose to start antiviral therapy with IFN in Naïve group and NA-treated group. Compared with those before IFN treatment, there was no noteworthy variation in the percentage of HLA-DR+CD38- CD4+ T lymphocytes in Naïve group and NA-treated group after IFN treatment . In both Naïve group and NA-treated group, HLA-DR+CD38dim CD4+ T lymphocytes proportion increased considerably after 4 weeks of IFN treatment . HLA-DR+CD38dim CD4+ T lymphocyte percentage in the Naïve group after 12-24 weeks of IFN treatment did not differ dramatically from that after 4 weeks of IFN treatment (P = 0.2020), but the HLA-DR+CD38dim CD4+ T lymphocyte percentage in NA-treated group was dramatically less than that in IFN treatment for 4 weeks (P = 0.0090). The change trend in HLA-DR+CD38hi CD4+ T lymphocyte percentage was consistent in Naïve group and NA-treated group during the treatment of IFN. At the beginning of treatment, HLA-DR+CD38hi CD4+ T lymphocyte percentage in both groups increased compared with the baseline, but there was no discernible variation . In the late stage of IFN treatment, HLA-DR+CD38hi CD4+ T lymphocyte percentage in both groups was dramatically higher than that at baseline . In Naïve group and NA-treated group, the proportion of HLA-DR+CD38- CD8+ T lymphocytes decreased gradually with the increase of IFN treatment time . In both Naïve group and NA-treated group, the proportion of HLA-DR+CD38dim CD8+ T lymphocytes increased notably after 4 weeks of IFN treatment . After 12-24 weeks of IFN treatment, the percentage of HLA-DR+CD38dim CD8+ T lymphocytes decreased notably compared with that of IFN treatment for 4 weeks in both groups . Moreover, with the increase of IFN treatment, the change trend of HLA-DR+CD38hi CD8+ T lymphocyte percentage in Naïve group and NA-treated group was similar, which increased significantly with the duration of treatment  (In order to further explore the dynamic changes in HLA-DR and CD38 coexpression subsets on CD4 0.0001) Table 2.+CD38- on CD4+ T lymphocytes in both groups during the treatment of IFN, but the proportion of CD8+ T lymphocytes decreased gradually. Except that the percentage of HLA-DR+CD38dim CD4+ T lymphocytes in Naïve group did not change significantly at the later stage of IFN treatment, the changes of other HLA-DR+CD38dim subsets on CD4+ and CD8+ T lymphocytes in both groups increased markedly first and then decreased gradually. Meanwhile, the proportion of HLA-DR+CD38hi on both CD4+ and CD8+ T lymphocytes increased significantly as the increase of IFN treatment time.The aggregate data showed that there was no significant change in HLA-DRP = 0.0010; P < 0.0001). At 12-24 weeks after IFN treatment, the level of HBsAg in NA-treated group declined markedly when compared to the baseline (P = 0.0270), but HBV DNA load had no significant change compared with the baseline (P = 0.3240). ALT and AST levels in Naïve group and NA-treated group were markedly higher than their respective baselines early in the course of IFN treatment , but there was no discernible change compared with the baseline in the late stage of IFN therapy , as shown in In Naïve group and NA-treated group, the HBsAg levels of all samples collected at 12-24 weeks after IFN treatment decreased more than 0.5 lg IU/mL compared with their respective treatment baselines. In Naïve group, compared with the baseline, HBsAg and HBV DNA levels declined considerably at 12-24 weeks after IFN treatment . HLA-DR+CD38- and HLA-DR+CD38dim subset proportions on CD4+ and CD8+ T lymphocytes were not linked with HBsAg level. Collectively, these findings suggested that when the patients did not enter the plateau phase, the proportion of HLA-DR+CD38hi CD4+ T lymphocytes and HLA-DR+CD38hi CD8+ T lymphocytes increased gradually with the decrease of HBsAg level during IFN treatment.Then, we used the longitudinal data collected from all patients in both groups to analyze the correlation between HBsAg and HLA-DR and CD38 coexpression subsets on CD4phocytes Figure 2+CD38hi subset on CD4+ and CD8+ T lymphocytes played a role in the plateau phase following long-term treatment of IFN, we continued to analyze the data of patients in the Plateau group after IFN intermittent treatment and IFN retreatment. Our data showed that after 12-24 weeks of IFN intermittent therapy, the proportion of HLA-DR+CD38- CD4+ T lymphocytes (P < 0.0001) and HLA-DR+CD38dim CD4+ T lymphocytes (P < 0.0001) increased significantly compared with the plateau baseline, while the HLA-DR+CD38hi CD4+ T lymphocyte percentage decreased significantly compared with the plateau baseline (P < 0.0001). After 12-24 weeks of IFN intermittent treatment, the corresponding subsets of CD8+ T lymphocytes also had a similar pattern. Compared with the plateau baseline, the proportion of HLA-DR+CD38- CD8+ T lymphocytes (P < 0.0001) and HLA-DR+CD38dim CD8+ T lymphocytes (P < 0.0001) increased significantly, while the proportion of HLA-DR+CD38hi CD8+ T lymphocytes decreased markedly (P < 0.0001) after IFN intermittent treatment with 12-24 weeks. In comparison to IFN intermittent treatment, the proportion of HLA-DR+CD38- CD4+ T lymphocytes and HLA-DR+CD38dim CD4+ T lymphocytes decreased markedly after 12-24 weeks of IFN retreatment . However, compared with plateau baseline, there were no noteworthy variation in both the proportion of HLA-DR+CD38- CD4+ T lymphocytes and HLA-DR+CD38dim CD4+ T lymphocytes after IFN retreatment . After 12-24 weeks of IFN retreatment, HLA-DR+CD38hi CD4+ T lymphocyte percentage was considerably more than that after IFN intermittent treatment (P = 0.0130), but dramatically lower than that at the plateau baseline (P = 0.0180). Likewise, HLA-DR+CD38- and HLA-DR+CD38dim subsets proportion on CD8+ T lymphocytes after 12-24 weeks of IFN retreatment was greatly lower than that after IFN intermittent treatment, respectively , but there was no discernible change compared with the plateau baseline . Furthermore, HLA-DR+CD38hi CD8+ T lymphocyte proportion increased considerably following IFN retreatment compared to IFN intermittent treatment. (P = 0.0060), but considerably lower than that at plateau baseline (P = 0.0160) (In order to further explore whether the persistent high proportion of HLA-DR 0.0160) Table 3.P > 0.9999). HBsAg level of each sample collected after IFN retreatment in Plateau group declined more than 0.5 lg IU/mL compared with that after IFN intermittent treatment, which was statistically noteworthy (P = 0.0030), and considerably lower than that at plateau baseline as well (P = 0.0060). During the IFN intermittent treatment and IFN retreatment, HBV DNA load in Plateau group had no significant change . In comparison to the baseline of plateau phase, ALT (P < 0.0001) and AST (P < 0.0001) levels in Plateau group decreased significantly after IFN intermittent treatment, while compared with those after IFN intermittent treatment, ALT (P < 0.0001) and AST levels(P < 0.0001) increased considerably in Plateau group after IFN retreatment, as shown in There was no significant variation in HBsAg level in Plateau group before and after IFN intermittent treatment  responses, while CD8+ T lymphocytes contributed to the generation of antiviral cytokines, such as IFN-γ and tumor necrosis factor-α, and could differentiate into CTL lymphocytes to exert cytotoxic activity to clear virus-infected hepatocytes (+ T and CD8+ T subsets in three groups of patients under different treatment background at the baseline, it could be found that for both CD4+ T and CD8+ T lymphocytes, long-term IFN treatment significantly decreased the proportion of HLA-DR+CD38- subsets and dramatically increased the proportion of HLA-DR+CD38hi subsets. However, the results of HLA-DR+CD38dim subsets in CD4+ and CD8+ T lymphocytes were slightly different. At this time, the dynamic changes of subsets in Naïve and NA-treated group during IFN treatment were analyzed, and it was found that the proportion of HLA-DR+CD38- CD8+ T lymphocytes decreased gradually during IFN treatment, but the change in HLA-DR+CD38- CD4+ T lymphocyte percentage during treatment was not significant. Actually, this abnormal phenomenon might be associated with the following reasons: it could be seen from the data of this study that the percentage change of each subgroup in CD4+ T lymphocytes was slighter than that in CD8+ T lymphocytes during IFN treatment. In the course of IFN treatment, HLA-DR+CD38- CD4+ T lymphocyte proportion in NA-treated group decreased gradually, although there was no statistical difference. In addition, it might also be related to the large dispersion of samples. In the course of IFN treatment, the change trend of HLA-DR+CD38dim subsets which played a positive function of virus clearance was found to be significantly increased at first, and then gradually decreased. This trend was most typical in CD8+ T lymphocytes, which was consistent with earlier stydies (+CD38dim CD4+ T lymphocyte percentage could be explained as the long-term treatment of IFN eventually led to the decrease of HLA-DR+CD38dim subgroup percentage on T lymphocytes, which was based on the increase of the percentage of this subgroup in the early stage of IFN treatment. At this time, reduced HLA-DR+CD38dim CD4+ T lymphocyte subsets percentage was still higher than that at baseline. The proportion of overactivated and exhausted HLA-DR+CD38hi subsets increased significantly during IFN treatment for both CD4+ and CD8+ T lymphocytes.CD4atocytes . Through stydies , 28. Dur+CD38hi subsets. With the extension of IFN treatment time, the proportion of HLA-DR+CD38hi subsets of both CD4+ and CD8+ T lymphocytes would gradually increase as the HBsAg level fell. In summary, our research showed that in the process of IFN treatment, the percentage of HLA-DR+CD38dim subsets that played a positive virus clearance function was significantly increased in the initial stage of IFN treatment, which was helpful to quickly eliminate the virus and reduce the level of HBsAg. At the same time, HLA-DR+CD38dim subsets gradually differentiated into HLA-DR+CD38hi subsets, which represented depletion. In the late course of IFN treatment, HLA-DR+CD38dim subset percentage decreased, while the percentage of HLA-DR+CD38hi subsets increased gradually.Moreover, HBsAg level in Naïve group and NA-treated group decreased significantly during IFN treatment. ALT and AST levels increased significantly in the initial stage of IFN treatment, which was linked with the enhancement of T lymphocyte immunological performance and NK lymphocyte activity after IFN treatment, indicating a better antiviral effect . The cor+ T lymphocytes (+CD38- and HLA-DR+CD38dim on CD4+ and CD8+ T lymphocytes elevated significantly, while HLA-DR+CD38hi subsets decreased significantly. After IFN retreatment with 12-24 weeks, HLA-DR+CD38- and HLA-DR+CD38dim subsets decreased significantly, while HLA-DR+CD38hi subsets increased significantly, which was consistent with the above demonstration. Before and after retreatment of IFN, the level of HBsAg declined significantly, while the level of ALT and AST elevated significantly, which also meant that IFN began to exert its antiviral effect and further enhance the function of immune lymphocytes (+CD38- and HLA-DR+CD38dim subsets on CD4+ and CD8+ T lymphocytes after IFN retreatment compared with the baseline of IFN plateau phase. At this time, the proportion of HLA-DR+CD38hi subsets on CD4+ and CD8+ T lymphocytes was dramatically lower than that at the baseline of plateau phase, respectively. In a word, the persistent high proportion of HLA-DR+CD38hi subsets on CD4+ and CD8+ T lymphocytes was related to the occurrence of IFN plateau phase. After IFN intermittent treatment, the proportion of HLA-DR+CD38hi subsets decreased, suggesting that the immune disorder of T lymphocyte overactivation may be restored. During the IFN retreatment, the proportion of HLA-DR+CD38hi subsets increased gradually, but it did not reach the higher level compared with the baseline of plateau phase. Therefore, IFN could continue to play an antiviral role and HBsAg began to decline again.The plateau phase occurred after long-term treatment of IFN, which meant that the antiviral effect of IFN could not be continued. Some studies have shown that it might be related to the gradual decrease of IFN receptor expression , the prophocytes . Whetherphocytes , 29. Mea+CD38dim and HLA-DR+CD38hi subsets on T lymphocytes could not be determined without further functional verification. Therefore, more evidence is needed to verify the role of HLA-DR and CD38 coexpression subsets on T lymphocytes when treating CHB using IFN.Our study had several drawbacks, including a small sample size and lack of enrollment of patients who participated in the whole period from initial IFN treatment to the IFN plateau stage. More importantly, the exact functional properties of HLA-DR+CD38hi subsets on T lymphocytes. IFN intermittent treatment could significantly reduce the proportion of HLA-DR+CD38hi subsets, helping regain the antiviral efficacy of IFN during IFN retreatment.In summary, our study demonstrated that the HLA-DR and CD38 coexpression subsets on T lymphocytes dynamically changed over the course of receiving long-term IFN treatment. The appearance of IFN plateau phase was related to the persistent high proportion of HLA-DRThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Beijing Ditan Hospital Affiliated with Capital University of Medical Sciences. The patients/participants provided their written informed consent to participate in this study.RH, ML and YX contributed to study concept and design. YLi, XB and LY conducted experiments and collected the data. YLi, GS, SX, HL, XB and LY collected the information of the patients. TJ, WD, SWa, LZ, YLu and YG ordered laboratory materials. YLi performed the statistical results and wrote the first draft. HH, SWu, RL, MC, MX, LH and XC edited the English version. All authors contributed to the article and approved the submitted version.