In South Korea, a total of 21 African swine fever (ASF) infected farms were confirmed during 2019–2021. ASF viruses (ASFVs) were isolated from the blood and spleen samples of the 21 affected farms and their genetic characteristics were analyzed. Phylogenetic analysis indicated that the 21 Korean ASFV strains belonged to p72 genotype II and serogroup 8. All isolates were of the intergenic region (IGR) II variant with 10 tandem repeat sequences between I73R and I329L and the central variable region (CVR) 1 variant of the B602L gene. There were no IGR variations between the A179L and A137R and between the MGF 505 9R and10R nor mutations in the O174L, K145R, MGF 505-5R, CP204L, and Bt/Sj regions. The genes of the 21 ASFV strains were identical to those of Georgia 2007/1 and Chinese and Vietnamese strains ; however, X69R of the J268L region of the 18th isolate (Korea/Pig/Goseong/2021) had three nucleotide (CTA) insertions at the 209th position, which led to the addition of one tyrosine (Y) at the C-terminal. This suggests that there are variations among ASFVs circulating in South Korea and the 18th ASFV-infected farm was due to a variant different from those of the other 20 pig farms. African swine fever (ASF) is one of the most fatal viral diseases affecting swine. ASF has high mortality and, due to the lack of effective vaccines and treatments, remains a huge threat to the pig industry and food security worldwide . With reOn 16 September 2019, the first ASF outbreak in South Korea was reported at a pig farm in Paju City, Gyeonggi Province. The index farm was located in the northwestern region near the border between South and North Korea. Until 9 October 2019, a total 14 ASF cases was confirmed in 4 cities/counties located in the northwestern border region . ASF hasAsfarviridae family [African swine fever virus (ASFV), the causative agent of ASF, is a large double-stranded DNA virus that is the only known member of the e family . Dependie family ,9. The Ae family . ASFV ste family ,12. ASFVe family .In a previous study , 14 ASFV2 incubator. The prepared samples were inoculated and 1% pig erythrocyte suspension was added. The virus in the cell supernatants was confirmed by OIE TaqMan quantitative polymerase chain reaction and the cyclic threshold (Ct) was subsequently estimated. Virus isolation was confirmed via hemadsorption (HAD).EDTA-treated whole blood or spleen samples were collected from 21 infected domestic pig farms between 2019–2021. Tissue samples were homogenized and centrifuged. The supernatant was filtered and inoculated into porcine alveolar macrophage (PAM) cells for virus isolation, according to the procedures of the Center for Animal Health Research, the European Union Reference Laboratory of ASF, and WOAH . BrieflyVirus antigens in PAM cells were also confirmed by fluorescent antibody test, according to the Diagnostic Manual of WOAH (2012). Four days after the first passage, the infected PAM cells were fixed in 80% acetone. Acetone was discarded and the plate was air-dried at room temperature. Anti-ASF VP72 monoclonal antibody  was incubated for 1 h at 37 °C. After being washed with PBS three times, cells were incubated in goat anti-mouse IgG − h + I conjugated with FITC . Fluorescence was observed and compared with mock infected PAM cells as a negative control.® RSC whole blood DNA kit for EDTA-treated whole blood and Maxwell® RSC viral total nucleic acid purification kit  for tissue homogenates, processed through the Maxwell® RSC 48 instrument, according to the manufacturer’s instructions. To genetically characterize the ASFV isolates, 12 gene markers were amplified and sequenced. First, we amplified the B646L gene encoding p72 for genotyping, and the EP402R encoding CD2v to classify serogroup, as previously reported [I73R-I329L) [A179L-A137R) [MGF 505 9R/10R) [Nucleic acid was extracted from the collected samples using Maxwellreported ,16,17. TR-I329L) , IGR betL-A137R) , IGR bet 9R/10R) ,20, CP20 9R/10R) , O174L,  9R/10R) , Bt/Sj,  9R/10R) . The PCRhttps://www.mbio.ncsu.edu/bioedit/bioedit.html accessed on 15 November 2020). Phylogenetic analysis of nucleotide sequences of the p72 genotype and the EP402R gene for serogroup were conducted using MEGA X 10.2 software (http://www.megasoftware.net accessed on 19 November 2020)All sequences of the 12 gene markers were analyzed using BioEdit 7.2 , MN603968 (E183L), MT335858 (EP402R), MN631140 (CVR), and MN603969 (IGRI73R-I329L); also, GenBank further contains the whole-genome sequence of Korea/Pig/Paju1/2019 (accession number MT748042). Phylogenetic trees of the p72 genotype and CD2v serogroups are shown in All 21 ASFV isolates were p72 genotype II, serogroup 8, CVR 1, and IGRThe p72 genotype II is the most dominant ASFV genotype worldwide and ASFVs of this genotype have been detected in all Asian countries. It should be noted that recently p72 genotype I viruses have been reported to be circulating in China in addition to genotype II . The EP4To obtain an enhanced resolution of the genetic characteristics of the ASFV isolates, 10 additional genes were analyzed. The CVR sequence of B602L was used to increase the intragenotypic discrimination. The CVR region is translated into amino acid sequence and grouped together. Overall, the 21 Korean isolates had a tandem amino acid repeat sequence (BNDBNDBNAA) within the CVR region, which corresponded to the p72 genotype II CVR1 . The GII-CVR1 profile was 100% identical to Georgia 2007/1, Pig/HLJ/2018, China/2018/AnhuiXCGQ, and VNUA/HY-ASF1. In Estonia, GII-CVR1 variants with single nucleotide polymorphism (SNP) 1 and GII-CVR2 deleted with 3 amino acid tetramer repeats (CASMCADTNVDT) were detected .I73R-I329L) is a genetic marker that can further discriminate between p72 genotype II viruses. Currently, four IGRI73R-I329L variants have been identified: IGRI73R-I329L I, IGRI73R-I329L II with 10 nucleotides (nt) of TRS in comparison to IGRI73R-I329L I, IGRI73R-I329L III containing additional 10 nt of TRS in comparison to IGRI73R-I329L II, and IGRI73R-I329L IV possessing extra 10 nt of TRS in comparison to IGRI73R-I329L III. The p72 genotype II IGRI73R-I329L II is the most frequently identified in type in Europe and Asia. IGRI73R-I329L I, III, and IV can be formed owing to the continuous circulation of ASFV in a pig population of a region. In South Korea, all ASFVs isolates from domestic pig farms in South Korea during 2019–2021 were IGRI73R-I329L II, while IGR I73R-I329L I, II, and III were detected in the wild boar population during surveillance in the northwestern border region in 2019 [I73R-I329L I, II, and III were found from wild boar in the northeastern border region (Jilin province) in 2018 and pig farms in the southern region in 2019, respectively [I73R-I329L I, II, and III were also reported in Vietnam [I73R-I329L I, II, III, and IV were detected [IGR between I73R and I329L  and between the MGF 505 9R and 10R (IGRMGF 505 9R/10R) are genetic markers used for subgrouping p72 genotype II viruses [A179L-A137R were found in southern Vietnam. One strain (ASFV/VN/Pig/Hanoi/02) was IGRI73R-I329L II, while the other (ASFV/VN/Pig/Hanoi/07) IGRI73R-I329L I [MGF 505 9R/10R variant into three types: MGF-1 without TRS insertion, MGF-2 with 17 nt of TRS (GATAGTAGTTCAGTTAA), and MGF-3 containing an additional 17 nt of TRS compared to MGF-2. MGF-2 was previously confirmed from seven Russian strains and nine Polish isolates [A179L-A137R and IGRMGF 505 9R/10R.The IGRs between the A179L and A137R . For K145R and MGF 505-5R, variants were divided into two groups. Variant I was 100% homologous to those of Georgia 2007/1, while variant II contained SNPs specific to Polish and Ukrainian isolates, which correspond to C65167A in K145R and G38332A in MGF 505-5R, respectively [In Eastern Europe, three genes, O174L, K145R, and MGF 505-5R, were used as molecular fingerprints to enhance intragenotypic resolution. In Poland, in-depth insight into the spatiotemporal clustering of ASFVs was obtained based on the analysis of these genes with IGRectively . None ofThe CP204L gene encoding the p30 and Bt/Sj region of the 21 Korean ASFV strains was identical to those of Georgia 2007/1, with no mutations. The J268L region located in the left variable region included ASF G ACD 00320, 00330, 00350, and X69R genes at 19436–20581 positions in the Georgia 2007/1 strain. There was no variation in ASF G ACD 00320, 00330, and 00350 genes in Korean ASFV strains, which were identical to those of Georgia 2007/1; however, X69R gene of the 18th isolate, Korea/Pig/Goseong/2021, possessed 3 nt (CTA) insertion at the 209th position, leading to the addition of one tyrosine (Y) at the C-terminal .I73R-I329L II, and CVR1. They did not contain TRS insertions in IGRA179L-A137R and IGRMGF 505 9R/10R; in addition, no variations in the O174L, K145R, MGF 505-5R, CP204L, and Bt/Sj regions were found among the 21 Korean isolates. The analyzed genes were identical to those of Georgia 2007/1, Chinese strains Pig/HLJ/2018 and China/2018/AnhuiXCGQ, and Vietnamese strain ASFV_NgheAn_2019; however, the X69R gene of the J268L region in the 18th isolate (Korea/Pig/Goseong/2021) possessed one tyrosine (Y) insertion at the 209th position. This suggests that there are slight variations of ASFVs circulating in South Korea and the source of the virus of 18th ASF-infected farm was due to a variant different from that in the other 20 pig farms.All 21 ASFV strains isolated from pig farms in South Korea during the 2019–2021 outbreaks belonged to p72 genotype II, serogroup 8 with IGR Analysis of whole-genome sequence of ASFV isolates can fully characterize the isolates. Though whole-genome sequencing itself can be over quickly by next-generation sequencing, subsequent genetic analysis is highly time-consuming. On the other hand, investigation of select gene markers of ASFV isolates by Sanger sequencing has important merit as it can be performed quicker to identify significant variations between ASFV isolates and allows for the rapid accumulation and sharing of data for specific genes, before the whole-genome sequence is eventually shared. To provide more comprehensive data on the viral genome, further studies are required to investigate other possible differences between isolates through analysis of other potential gene markers and whole-genome sequencing.


JAZF1 rs864745 A>G variant and type 2 diabetes (T2D), type 1 diabetes (T1D) risk, and their correlation with clinical features, including islet function, islet autoimmunity, and plasma lipid levels.This study aims to reveal the association between JAZF1 and T2D, T1D, and islet-specific autoantibody status under the additive model, while multiple linear regression was used to assess its effect on glycemic-related quantitative traits and plasma lipid levels.We included 2505 healthy controls based on oral glucose tolerance test (OGTT), 1736 unrelated T2D, and 1003 unrelated autoantibody-positive T1D individuals. Binary logistic regression was performed to evaluate the relationships between rs864745 in JAZF1 and T2D, T1D, or their subgroups (All P > 0.05). For glycemic traits, we found that the G allele of this variant was significantly associated with higher 120 min insulin level, insulinogenic index (IGI), corrected insulin response (CIR), and acute insulin response (BIGTT-AIR)  in healthy individuals. Similar associations were observed in newly diagnosed T2D but not T1D individuals. Although this variant had no impact on islet autoimmunity (All P > 0.05), significant associations with plasma total cholesterol (TC) and low-density lipoprotein (LDL) level stratified by JAZF1 rs864745 variant were observed in the disease status of T2D (P = 0.002 and 0.003) and T1D (P = 0.024 and 0.009), with significant heterogeneity to healthy individuals.We did not find any association between rs864745 in JAZF1 rs864745 variant contributes to islet function and lipid metabolism, which might be put into genetic risk scores to assess the risk of related clinical features.The common  JAZF Zinc Finger 1 (JAZF1), a multifunctional regulatory factor, was initially identified as an orphan nuclear receptor corepressor and involved in gluconeogenesis and lipid metabolism. The human JAZF1 gene, located on chromosome 7p15.2, is predominantly expressed in insulin-responsive organs such as the liver, fat, skeletal muscle, and pancreas (JAZF1 is expressed in both human (JAZF1 is an important regulator of endoplasmic reticulum (ER) stress and ribosome biogenesis via a feedback action preventing the activation of ER and p53 stress-mediated β-cell apoptosis  (Genome-wide association studies (GWAS) have shown that the risk A allele of rs864745, an intron variant in  1 (T1D)  in Europ 1 (T1D)  in vario 1 (T1D) , 14–17.  1 (T1D) .JAZF1 and T1D, T2D subgroups, and also β-cell function and lipid metabolism in both healthy and diabetes disease status.Studies have demonstrated that T1D and T2D have heterogeneous characteristics in terms of both genetic , 19 and This study includes unrelated 1736 T2D, 1003 T1D, and 2505 non-diabetic controls. Their clinical characteristics are shown in All non-diabetic controls and T2D individuals measured plasma glucose and serum insulin levels at fasting, 30, and 120 min after OGTT. Serum C-peptide levels were measured in newly diagnosed T1D patients (disease duration less than 3 months) on fasting, 30, 60, 120, and 180 min standard mixed meal tolerance test (MMTT). Serum insulin levels were measured by an insulin radioimmunoassay kit . Serum C-peptide levels and plasma lipid levels, including high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), total cholesterol (TC), and triglyceride (TG), were determined by the chemiluminescence method . The methods for detecting islet-specific autoantibodies were described in our previous studies .JAZF1 rs864745 variant was performed using the Sequenom Massarray . The primer sequences and UEP primers were as follows: Forward, 5-ACGTTGGATGCATTGAACATTTCCTACAACC-3; Reverse, 5-ACGTTGGATGCCATATAAGTGATGCTCAAA-3; UEP, 5-ATGCTCAAATATAATTTGAACTGTTA-3. The genotyping success rate was above 98%, and 100 randomly selected samples were repeated for complete agreement. The genotype distribution in control, T2D, and T1D groups were consistent with Hardy-Weinberg equilibrium (P > 0.05).Genomic DNA was extracted from peripheral blood lymphocytes using the QIAamp DNA Blood Extraction Kit . Genotyping of the JAZF1 and T2D and T1D subgroups, and islet-specific autoantibody status. The effect of this variant on glycemic quantitative traits and serum lipid levels were analyzed by multiple linear regression. The glycemic quantitative traits were adjusted by sex, age, and BMI except that BIGTT-AIR and BIGTT-SI were corrected for age. Serum lipid levels were also adjusted by lipids medication in addition to age, gender, and BMI. All P-values were two-sided, and P < 0.05 was considered significant. The heterogeneity was considered significant with 2I > 75% and P < 0.05. Statistical analyses were performed using SPSS 18.0 and STATA 11.0.We measured and calculated insulin release and sensitivity derived from the OGTT in non-diabetic controls and T2D individuals according to the formulas described previously, including homeostasis model assessment of β-cell function (HOMA-B) and insulin resistance (HOMA-IR), insulinogenic index (IGI), acute insulin response index (BIGTT-AIR), insulin sensitivity index (BIGTT-SI), corrected insulin response (CIR), and Matsuda’s insulin sensitivity index (Matsuda ISI) . Serum iJAZF1 rs864745 A>G variant and T2D or T1D risk in total (P > 0.05). Although obesity is a critical risk factor for T2D, no association with this variant was observed in lean or obese T2D subgroups (P > 0.05), stratified by BMI status according to the Chinese criteria  individuals, as shown in We analyzed OGTT-derived indicators of glycemic-related traits in healthy individuals. As shown in We did not find any association between this variant and plasma lipid levels  in the healthy individuals based on OGTT (All P > 0.05). However, the G allele of this variant was associated with lower plasma LDL and TC levels in both T2D (P = 0.003 and 0.002) and T1D status (P = 0.009 and 0.024). Compared with healthy controls, their impacts on LDL and TC levels had significant heterogeneity in T2D (P = 0.049 and 0.030) and T1D (P = 0.017 and 0.034) status, as shown in JAZF1 region was reported as the genome-wide significant region in T1D , demonstrated that this variant affected JAZF1 gene expression in multi tissues, including the liver, skeletal muscle, and pancreas. Thus, we speculated that two distinct mechanisms for this variant are involved in protecting individuals from diabetes. One is by improving β-cell function, and the other is modulating lipid metabolism (via decreasing TC and LDL levels) in the hyperglycemia state.Studies have reported that dyslipidemia is a well-established risk factor for T1D and T2D –39. JAZFs in T2D . The benipocytes , 40–42. d traits . Of thesJAZF1 rs864745 variant is linked to the improvement of β-cell function as well as lipid metabolism. Considering the limitation of the reproducibility with a single variant, more T1D or T2D genetic risk loci should be further studied for clinical subgroups and phenotypic of T1D and T2D patients in diabetes screening and precision therapy.In conclusion, we found that the The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee from the First Affiliated Hospital of Nanjing Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.HD performed statistical analyses and interpretation of data and drafted the initial manuscript. HL, YQ, and LJ were responsible for the analyses and interpretation of data. HJ, MS, and HC contributed to laboratory measurements. YC and SZ contributed to the collection and selection of samples. QF and TY gave a critical revision of the manuscript. KX directed the study design and provided a critical revision of the manuscript. All the co-authors gave the final approval of the version.This study was supported by grants from the National Natural Science Foundation of China , and the Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University (PY2021005).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified.In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes.in vitro.In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development. Moreover, a positive correlation was demonstrated between the rates of evolution in HIV-1 and disease progression (Human immunodeficiency virus type 1 (HIV-1) is one of the potential pathogenic exogenous retroviruses, which is quickly evolving into new subtypes driven by the high frequency of mutation, recombination, and replication . Therefogression . Hence, The HIV-1 envelop (Env) protein, a trimer of gp41/gp120 heterodimers, exhibits 30 ± 3 potential N-linked glycosylation sites (PNGSs) per protomer , and it Among the demarcated five variable loops in HIV-1 gp120, the V3 loop has been demonstrated as a conserved region with amino acid variation limited to ~20% of the loop’s residues , essentiin vitro  specifically recognize the paratope of VRC-01 bNAb and compete with gp120 protein for binding to VRC-01 bNAb. These proteins, called VRA binders, mimic natural epitopes in the CD4 binding region of gp120 protein, and when used as immunogens, they stimulate the production of anti-gp120 antibodies. Hyperimmune mouse sera show the neutralization of HIV-1 pseudoviruses of Clades B and C in vitro . This apin vitro , was usein vitro . Elicitein vitro . As glycThe combinatorial library of Myomedin variants was designed and assembled using a series of polymerase chain reactions (PCR) by Phusion High-Fidelity DNA Polymerase , as reported earlier .https://www.hivreagentprogram.org). Both the PGT121 and PGT126 IgG1 λ type were employed as target proteins for ribosome display (RD) selection and in the enzyme-linked immunosorbent assay (ELISA) applications. Human IgG1 with λ light chain purchased from Sigma-Aldrich, St. Louis, MO, was utilized as an isotype control in the preselection of the library during RD as well as an isotype control in ELISA. All the other secondary antibodies of molecular grade were used in this study.Broadly neutralizing anti-HIV-1 gp120 monoclonal antibodies PGT121 (cat# ARP-12343) and PGT126 (cat# ARP-12344) were acquired from the NIH AIDS Reagent Program  of human IgG1 λ type and varied concentrations  of PGT121 or PGT126 bNAbs in 100 mM bicarbonate/carbonate buffer (pH 9.6) were used to conduct the preselection and adjust the stringency during each round of RD selection, respectively. To increase the stringency during each round of RD selection, different concentrations of TWEEN 20 in phosphate buffered (pH 7.2), i.e., 0.05% Tween/5 times, 0.05% Tween/10 times, and 0.25% Tween/10 times were used in the first, second, and third cycle, respectively. Following, the complementary DNA (cDNA) collected from the third round of RD selection was amplified into double-stranded DNA using PCR with primers JOIN-F(CTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAAAGCGAGCTGGCCG) and JOIN-R (GAACCGACCGCGGATCCACCCTGTTTACGAATCCATTCTT), and the resulting product was further amplified with primers His-Myo-F (CAGTCCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCAAAAGCGAGCTGGCCG) and JOIN-R to insert cloning sites, as reported earlier  cells cultured in Luria-Bertani (LB) broth with kanamycin (60 µg ml-1). Briefly, the bacteria culture was incubated at 37°C to reach the optical density (OD600) ~0.8 under continuous shaking conditions (230 rpm), followed by adding 1 mM IPTG for protein production at 32°C for the next 4 h on shaking conditions. Later, the cells were harvested by centrifugation  and suspended in TN buffer , disrupted using a sonicator (Misonix Sonicator® 3000 Ultrasonic Cell Disruptor), and the supernatant containing soluble protein was collected by centrifugation . The cell lysates were then analysed and used for protein purification on the Ni-NTA agarose column. Myomedin variants containing C-terminal Avi-tag sequence consensus were also produced (His6-Myomedin-Avi) in E. coli BL21 (DE3) BirA strain cultivated in LB broth with 50 µM d-biotin , kanamycin (60 µg ml-1), and chloramphenicol (30 µg ml-1).The selected Myomedin proteins (~16 kDa) containing hexahistidine (His6)-tag at the N-terminus and V5-tag at the C-terminus (His6-Myomedin-V5) were expressed in -1 of PGT121 IgG1, PGT126 IgG1, or IgG1 λ diluted in 100 mM bicarbonate/carbonate solution, pH 9.6, were immobilized in 96-well PolySorp plates (Thermo Scientific™ Clear Flat-Bottom Immuno Nonsterile 96-Well Plates) at 4°C overnight. In the following steps, the plates were washed three times with PBS (pH 7.4) containing 0.05% Tween 20 solution (PBST), and wells were blocked with PBST with 1% BSA (PBSTB) for 2 h at room temperature (RT). Then, the cell lysate samples or purified Myomedin proteins diluted in PBSTB were incubated in a 96-well plate for 1 h and later detected for binding with coated antibody using anti-V5-tag–HRP  in PBSTB (1:10 000 dilution). For competition assay, multimerizing HIV-1 consensus B gp120 protein prepared and characterized in detail previously (-1) and incubated in the 96-well plate for 1 h at RT. Then, the Myomedin variants were detected by anti-Flag tag antibody  or by streptavidin–HRP conjugate  in the case of in vivo biotinylated Avi-tagged variants. In both ELISA setups, results were observed as the reaction of HRP with TMB -Complete 2 substrate , which was stopped by 2 M sulfuric acid. For binding or competition assay, the absorbance at 450 nm was measured using the Multi-Mode ELISA Reader (BioTek Synergy™ HTX system).ELISA was used to screen the Myomedin variants binding with the selected bNAbs compared to isotype control (human IgG1 λ type). Herein, 5 µg mleviously –29 was sThe structure of Myomedin-derived MLD proteins was modeled using the MODELLER 9v14 suite of programs  based onExperimental female BALB/c mice of weight 18-22 g and 6-8 weeks old , housed following ARRIVE guidelines , were usThe binding of serum antibodies targeting HIV-1 Env was determined using multimerizing HIV-1 consensus B gp120 protein –29 and aELISA plates coated with 50 ng/well of multimerizing HIV-1 consensus B gp120 –29 proteIn individual Epp tubes PGT121 or PGT126 antibodies were serially diluted in blocking buffer, mixed with 100 ng of gp120 and 2 µl of individual serum in total volume of 200 µl, incubated at room temperature for 2 hours and then applied onto gp120-coated and blocked ELISA plates. After overnight incubation at 4°C, plates were washed, and bound mouse antibodies were detected as in previous section.The assay was performed following the protocol described previously , 38 utilAnalysis of variance (ANOVA), Kruskal-Wallis test and Dunn´s post-test were used to determine the statistical significance of differences between groups by statistical packages SPSSv.21 GraphPad Prism 5 Software  or .To verify the ability of Myomedin scaffold proteins to mimic glycan epitopes as a critical part of high-mannose patches on gp120 recognized by HIV-1 broadly neutralizing antibodies, two bNAbs, i.e., PGT121 and PGT126, were chosen as molecular targets. Monoclonal antibody PGT121 belongs to a group of super-candidate anti-HIV-1 neutralizing antibodies due to its potential to neutralize a wide portfolio of HIV viruses of several clades with high efficacy. Independently, PGT126 as a suitable representative of the PGT128 sub-family was selected as another V3 loop glycan targeting bNAb with high coverage of HIV-1 variants. Both antibodies were used as targets for the selection of Myomedin proteins using 3-round RD. Large-scale screening of bacterial lysates of individual Myomedin variants preferentially binding to the particular immobilized bNAb resulted in the identification of three variants of anti-PGT121 proteins called MLD033, MLD068 and MLD108, and three variants of anti-PGT126 Myomedins called MLB036, MLB041 and MLB049 Figure 210.5281/zenodo.6913156.We analyzed interactions of PGT121-124 and PGT125-128 subfamilies based on available structural data. The first subfamily can be represented by the selection of PDB structures containing Env/PGT121-precursor complex 5cez , glycan-To verify the most probable antibody binding areas for all selected MLB and MLD binders, we performed a flexible side chain global docking of corresponding proteins using ClusPro. Docking results are presented in Myomedin variants, MLB036, MLB041, MLB049, MLD033, MLD068, MLD108, and parental MyoWT, were injected as antigens into mice by intradermal route with Freund’s adjuvant by four series of immunizations as presented in -1 and 0.25 µg ml-1 inhibited the reactivity of MLB- and MLD-elicited hyperimmune sera. When irrelevant VRC01 bNAb was used, no competition was observed even at the concentration of 10 µg ml-1. Naive sera and MyoWT-immunized mouse sera did not titrate with PGT126 and PGT121  residue followed by an introduction of Serine (S) or Threonine (T) residue at the 336 position, to produce a new NXS/T sequence motif for the N-glycosylation at the N334 site  neutralized 70% of viruses, while the middle range concentration (0.1-1 µg ml-1) neutralized 57% of viruses and the lowest one (≤ 0.1 µg ml-1) only 44% viruses. Compared with this published coverage, MLD108-elicited sera neutralized 9 of 22 pseudoviruses (40.90%). Similarly, PGT126 in the concentration range of 1-50 µg ml-1 neutralized 60% of viruses, 0.1-1 µg ml-1 neutralized 50% of viruses and below 0.1 µg ml-1 neutralized only 40% of viruses  share sequence identity between MLB041 and MLB049, but their neutralizing potential found in hyperimmune murine sera mainly differs in Clade A, B and C pseudoviruses Figure 6in vitro identification of proteins binding selectively bNAb from highly complex combinatorial library to identify a set of binders for the selected bNAb. Binders were subsequently tested in experimental animals as the antigens for elicitation of gp120 binding and HIV-1 pseudoviruses neutralizing sera. Although we opted capturing the tested mAb on ELISA plates during binders' identification, we were aware of potential conformational changes in the bound mAb. Therefore, we used several competition assays to test the mAb-to-binder, Ab-to-gp120, and, after immunization, also hyperimmune serum-to-binder and serum-to-gp120 interaction in soluble conditions, which is important for antibody interaction with its target. Because during the neutralization process in vivo the target Env protein is a part of virus membrane, final analysis was focused on testing the sera neutralizing properties utilizing HIV-1-pseudotyped virus assay. Finally, we performed structural modeling of the interaction of best in vitro and in vivo acting binders with mAb using available crystal structures to, at least partially, address the nature of binders to mAb interaction. Our “non cognate antigen” approach is an alternative to strategies of structure-based reverse vaccinology (SBRV) identifying gp120-derived antigen for immunization by detail analyses of the pair mAb-recombinant gp120  which did not succeed to provide a construct for the generation of successful broadly effective preventative vaccine. The aspects potentially associated with the failure of SBRV are discussed in detail by van Regenmortel  can be found in the article/The animal study was reviewed and approved by The Ethics Committee of the Faculty of Medicine and Dentistry  and the Ministry of Education, Youth and Sports, Czech Republic (MSMT-9487/2019-3).in silico modeling of Myomedin proteins interactions. PK and MKr performed the immunological analyses. PK, LRK, and MR performed immunization experiments. EV and LRK engineered and isolated recombinant gp120 proteins and isolated plasmids for pseudoviruses preparation. PK performed virus-neutralizing assays. PM and MR supervised the study. PM, MR, JČ, PK, and SB wrote the manuscript with input from all authors. All authors have read and agreed to publish this version of the manuscript.PM and MR conceived and designed the study. VDL, MK, and TZ performed a large-scale screening of Myomedin library and selection of MLB and MLD variants. HP, JŠ, and MM performed protein characterization assays. JČ performed


B and OBIC) in the presence or absence of anti-HBs were analyzed extensively in this study. Methodology. The amino acid substitutions of envelope proteins of 21 OBI strains, including 4 HBs (+) OBIB, 6 HBs (−) OBIB, 6 HBs (+) OBIc, and 5 HBs (−) OBIC samples, were analyzed and fully compared among groups of HBV genotypes and the presence of anti-HBs.  The coexistence of hepatitis B virus (HBV) and hepatitis B surface antibodies (anti-HBs) in occult hepatitis B virus infection (OBI) is a contradictory phenomenon, and the underlying mechanism is not fully understood. The characteristics of pre-S/S mutations in OBI genotypes B and C (OBIC were significantly higher than wild-type HBV (wt-HBV) genotype C strains, but only the mutation rate of S protein in OBIB was significantly higher compared to wild-type HBV genotype B. The mutation rates in S protein of anti-HBs (−) OBI were higher than anti-HBs(+) OBI samples . For these high-frequency substitutions in the pre-S/S region, the mutations sN40S and sK122R were found in 27.3% and 45.5% of anti-HBs (−) OBI strains, respectively. 7 substitutions were uniquely found in OBIC strains and 9 substitutions were commonly detected in OBIB and OBIC strains.  The mutation rates in pre-S1, pre-S2, and S proteins of OBI These results suggested that the mutations might occur randomly and were not selected by antibody pressure. Hepatitis B virus (HBV) infection is a major threat to human health worldwide, and nearly 2.57 billion people worldwide are estimated to be infected with HBV. China is a higher intermediate prevalence area of HBV. The prevalence of HBsAg for populations aged 1∼59 years is 7.18%. It is estimated that there are more than 93 million HBV-infected individuals in China, which results in a public health issue [OBI can be grouped into two types: seropositive OBI [hepatitis B core antibody (anti-HBc) and/or anti-hepatitis B surface antibody (anti-HBs) positive] and seronegative OBI (anti-HBc and anti-HBs negative) . The antIn general, the level of anti-HBs is low in OBI patients; for example, 4 of 120 HBsAg(−) healthcare workers with low <10 IU/L) and moderate levels (>10 to <100 IU/L) of anti-HBs were positive for HBV DNA examined by sensitive real-time PCR . It seem IU/L andThe mechanisms behind the occurrence of OBI associated with anti-HBs pressure are not fully understood. In this study, the pre-S/S mutations in OBI blood donors associated with anti-HBs elicited by vaccination or the host immune response to HBV were analyzed extensively, providing new data for the coexistence of HBV anti-HBs and even the mechanisms of OBI.136425 blood samples were collected by the Shaanxi Blood Center from January to October 2015. Blood samples were individually screened for anti-HIV, anti-HCV, HBsAg, and anti-TP by enzyme immunoassays (EIA) using two different reagents for two rounds . The level of alanine aminotransferase (ALT) was detected by the rate method .µL) of the Roche COBAS S 201 Nucleic Acid Detection System . The reactive pools were identified according to the single sample mode. All HBsAg (−)/HBV DNA (+) samples were confirmed and quantified by electrochemiluminescence immunoassay (ECLIA) using a reagent kit from Wantai . The study protocol was approved by the Ethics Committee of the Shaanxi Blood Center.HCV, HIV, and HBV nucleic acids were detected in 6 samples mixed-mode . The minimal detection limit for HBV DNA is 10 IU/mL, and the linear range of the standard curve is from 20 IU/mL to 2.0 × 10The Pre-S/S (1434 bp)/S (480 bp) region of the HBV genome was amplified by nested PCR according to a previous study . HBV DNAP1:5′-ACATACTCTTTGGAAGGCKG-3′; R1:5′-CGTCAGCAAACACTTGGC3′; P2:5′GCCTCATTTTGYGGGTCA-3′; R2:5′-AGCAAARCCCAAAAGACC-3′. The two sets of PCR primers for the S gene were: S1:5′-CTCGTGTTACAGGCGGGGTTTTTC-3′; R1:5′-CATCATCCATATAGCTGAAAGCCAA ACA-3′; S2:5′-TTGTTGACAAGAATCCTCACAATACC-3′; R2:5′-GCCCTACGAACCACTGAACAA ATGG-3′. The products were purified and sequenced by Jinweizhi Biotechnology Co., Ltd. using ABI 3730 DNA sequencer (Applied Biosystems).In the Kimura-2-parameter model with 1000 bootstrap replicates, phylogenetic trees were constructed using Mega-X software using the neighbor-joining method. The HBV reference sequences for multiple genotypes (A to H) were obtained from the National Center for Biotechnology Information genotyping tool. The deduced amino acid sequences of the pre-S/S regions  of OBI were compared to the corresponding genotype B or C consensus sequences of HBV wild-type strains. The control HBV wild-type strains were all Chinese strains obtained from the GenBank database. Accession number of genotype B were FJ386582, FJ386600, FJ386608, FJ386610, FJ386634, FJ386654, FJ386655, FJ386658, FJ386668, FJ386669, FJ386680, FJ386681, FJ386683, FJ386684, FJ386688; accession number of genotype C were FJ386577, FJ386579, FJ386585, FJ386587, FJ386603, FJ386604, FJ386614, FJ386619, FJ386639, FJ386644, FJ386649, FJ386657, FJ386661, FJ386662, FJ386685.t-test or the chi-square test, as appropriate. A difference with P < 0.05  was statistically significant.The SPSS software 18.0  was employed for statistical analysis. Group results were compared using a Of the 136425 sera samples, 95 were verified as HBsAg (−)/HBV DNA (+), accounting for 0.0696% of the investigated blood donors. These samples can be classified into 6 different groups based on the serological index . 10 sampThe pre-S/S region of HBV was amplified in 11 samples, including 8 anti-HBs (+) and 3 anti-HBs (−).The S region was amplified in those samples that failed to detect the pre-S/S region, and 10 samples, including 2 anti-HBs (+) and 8 anti-HBs (−), were amplified successfully. Finally, the pre-S/S genetic regions were successfully amplified in 21 of 85 OBI samples (24.71%). HBV genotypes were classified.B and OBIC, respectively (B and OBIC (2 : 8 vs. 2 : 9). The age, ALT levels, and viral load were various between genotypes B and C, but no significant differences were found between the two genotypes (P = 0.41–0.85).Based on phylogenetic analyses of OBI S region sequences. 10 and 11 strains were classified as OBIectively . The ratB and 11 OBIC strains, were aligned with the corresponding consensus sequences of wt-HBV strains (15 genotype B and 15 genotype C), and the results of the comparison were presented (C were significantly higher (P < 0.01). However, the pre-S1 and pre-S2 regions appeared well conserved between OBIB and genotype B wt-HBV strains , only the mutation rate in S proteins of OBIB was significantly higher (P < 0.01) than that of wt-HBV strains. When comparing the sequences of OBIc with OBIB, the substitution rate in the pre-S1 region was significantly higher in OBIC than in OBIB (P < 0.05), but the substitution rates in pre-S2 and S showed no statistical significance .Deduced amino acid sequences of envelope proteins obtained from OBI samples, including 11 pre-S/S and 10 S region sequences from 10 OBIresented . CompareC samples was statistically higher than that in anti-HBs (+) OBIC samples , but no statistical difference was found in anti-HB (−) OBIB and anti-HB (+) OBIB samples . The substitution rate in pre-S1 and pre-S2 proteins between groups of anti-HBs (−) and anti-HBs (+) in either OBIc or OBIB carriers appeared with no statistical significance (P > 0.05). The overall mutation rate considering Pre-S/S in OBIc strains was higher than in OBIB strains. However, for anti-HBs (−) OBI strains, only the mutation rate in S protein presented a statistical difference (P=0.046) between genotype B and C, whereas only the mutation rate in pre-S1 protein presented a statistical difference for anti-HBs (+) OBI strains between genotype B and C (P=0.01).Site mutations in the pre-S/S region of HBV strains from OBI carriers in the presence or absence of anti-HBs were aligned together . The difα” determinant were analyzed. The mutation rate of MHR in anti-HBs (−) OBIc strains was significantly higher than that in anti-HBs (+) OBIc strains , and the mutation rate of MHR in anti-HBs (−) OBIC strains was higher than that in anti-HBs (−) OBIB strains . Nevertheless, the mutation rates in the “α” determinant among different anti-HBs status and genotypes presented no statistical difference.Furthermore, amino acid substitutions in the major hydrophilic region (MHR) and the “B carriers, and sK122R was uniquely found in 45.5% of anti-HBs (−) OBI preferentially in OBIC strains. The substitutions ps1A60V, ps1G73S, ps1V90A, ps2I42T, sT118R/V/M, sI126T/M, and sD144E/N/A were uniquely found in OBIC strains in both anti-HBs (+) and anti-HBs (−) populations. The substitutions sV/T47K/A/E, sM133T/L, sF134I/L/R, sK160R/S/K, sV168A, sS174N, sL175S, and sV177A were commonly detected in OBIB and OBIC strains in both anti-HBs (+) and anti-HBs (−) populations.The high frequency of pre-S/S substitutions was classified as preferentially associated with either the anti-HBs (+) or anti-HBs (−) OBI strains considering different genotypes . The subOBI is a worldwide health problem, and the reported prevalence significantly varies depending on locations, populations, and the sensitivity of diagnostic assays. The detection rate of OBI in blood donors was 1 : 1436 in our study, which was approximately two times lower than that in the data from South China reported by the National Institute of Diagnostics and Vaccine Development in Infectious Diseases of China (1 : 631)  but was P > 0.05; 6.81% vs. 3.47% in genotype C, P < 0.05). Controversially, Wang JW et al. reported that the number and frequency of site substitutions in the S protein from anti-HBs (+) OBIC strains were higher than those associated with anti-HBs (−) OBIC strains, which suggested that the presence of anti-HBs might more effectively select variants in the S regions in genotype C than in genotype B OBI strains [The distribution of HBV genotypes presents clear differences in geographical regions, ethnicities, and clinical outcomes . Genotypα” determinant is a relatively conserved region within MHR, which serves as the most important immunodominant region in all HBV strains and is essential to the detection of HBsAg and development of HBV vaccines [Unlike HCV and some other hepatitis viruses, their antibodies could not provide protection and were the only evidence of infection. The anti-HBs were the neutralizing antibodies for HBV and the markers of recovery from acute infection or immunity from vaccination. The coexistence of HBsAg and anti-HBs seemed contradictory, but it was reported in many populations of different ages, sex, and immune situations , 6–8. Thvaccines . Mutatiovaccines –24. (2) vaccines .C strains obtained from blood donors with anti-HBs positive or negative. The functions of these mutations should be further studied. 13 individual substitutions in the sequences of the S region were found with high frequency (9.1∼45.5%) in OBI strains  and anti-HBs (−) OBI strains [In this study, ps1A60V, ps1G73S, ps1V90A in pre-S1 and ps2I42T in pre-S2 regions were only detected in OBI strains . The mutn of HBV , and sK1C strains with low mutation frequency, suggesting that this classical mutation might be better detected due to the improvement in HBsAg detection reagents, and the mutation may occur randomly regardless of the pressure of anti-HBs.Several substitution sites were detected in this study, including sN40S, sY100S/C sK122R, sI126T/M, sQ129R/L, sM133T/L, sF134I/L/R, sK160R/S/K, sD144E/N/A, and sV177A, have been reported to be associated with OBI . The claB and OBIC) and serological groups (anti-HBs positive and anti-HBs negative). The amino acid substitutions occurred more frequently in OBI than in wt-HBV, providing possible explanations for the occurrence and existence of OBI. The amino acid mutation rate in the S protein of anti-HBs (−) OBI samples was higher than anti-HBs (+) OBI samples, suggesting that the mutations in the S region are not selected by anti-HBs pressure resulting from vaccination or a natural host immune response to HBV. Our study provided insights into the underlying mechanisms by which the OBI occurs and the anti-HBs present in OBI carriers. However, the numbers of OBI strains that could amplify the pre-S/S region were small, and all the samples came from Shaanxi. The source of HBV strains was relatively single. Further work should be conducted based on a multicenter and large sample size, and more accurate results could be obtained. Furthermore, functional analysis of relevant genetic variants should be performed to elucidate the mechanism of OBI with anti-HBs positive in the future.The mutations in the pre-S/S region of OBI strains were analyzed and compared among different HBV genotype groups (OBI


Congenital hydrocephalus (CH) is a common neurological disorder affecting many newborns. Imbalanced neurogenesis is a major cause of CH. Multiple CH-associated mutations are within the RNA-binding domain of Trim71, a conserved, stem cell–specific RNA-binding protein. How these mutations alter stem cell fate is unclear. Here, we show that the CH-associated mutations R595H and R783H in Trim71 accelerate differentiation and enhance neural lineage commitment in mouse embryonic stem cells (mESCs), and reduce binding to mRNAs targeted by wild-type Trim71, consistent with previous reports. Unexpectedly, however, each mutant binds an ectopic and distinct repertoire of target mRNAs. R595H-Trim71, but not R783H-Trim71 nor wild-type Trim71, binds the mRNA encoding β-catenin and represses its translation. Increasing β-catenin by overexpression or treatment with a Wnt agonist specifically restores differentiation of R595H-Trim71 mESCs. These results suggest that Trim71 mutations give rise to unique gain-of-function pathological mechanisms in CH. Further, our studies suggest that disruption of the Wnt/β-catenin signaling pathway can be used to stratify disease etiology and develop precision medicine approaches for CH. This study shows that two congenital hydrocephalus (CH)-associated mutations in Trim71 accelerate mouse embryonic stem cells differentiation into neurons; while these mutations reduce binding to known Trim71 target mRNAs, the mutant Trim71s bind new and distinct targets, leading to gain-of-function effects that may contribute to the etiology of CH. Congenital hydrocephalus (CH) is a common birth defect affecting approximately 1 in every 1,000 newborns  and doesHuman exome sequencing studies identified multiple CH-associated mutations in several genes that are specifically expressed in embryonic and neural stem cells ,4. TheseTrim71 , sug, sug4],  defects . The CH- defects . HoweverCtnnb1 mRNA, which encodes β-catenin. This repression leads to decreased Wnt/β-catenin signaling activity specifically in R595H-Trim71 mutant mESCs. Increasing β-catenin levels alleviates the differentiation defects in the R595H-Trim71 mESCs, but not in R783H mESCs. These data suggest that gain-of-function mutations in the RNA-binding domain of Trim71 cause CH via distinct pathological mechanisms, that CH patients could be stratified based on these distinct mechanisms, and that precise therapeutic strategies could be designed for genetically diverse etiologies of CH.Here, we found that although R783H-Trim71 and R595H-Trim71 lead to premature differentiation and accelerated neural lineage commitment in mouse embryonic stem cells (mESCs), each Trim71 mutant binds a distinct and ectopic subset of target mRNAs. We discovered that R595H-Trim71, but not R783H-Trim71 nor WT-Trim71, specifically binds and represses the translation of We used mESCs as a model for mechanistic studies of the CH-associated mutations in Trim71 because the pathogenesis caused by Trim71 mutants is conserved between mouse and human , and TriThe protein levels of pluripotency factors  and coloCH is a neurological disorder that can be caused by impaired neurogenesis ,14. We fAltogether, these observations indicate that the two different CH-associated mutations in Trim71 lead to similar defects in embryonic stem cell differentiation and neural lineage commitment. However, our data also argue that these CH-associated mutations are gain-of-function mutations, leading to differentiation phenotypes that are not necessarily recapitulated by Trim71 KO.Both of the CH-associated mutations are located in the RNA-binding domain of Trim71. We recently showed that R783H-Trim71 and WT-Trim71 bind distinct subsets of mRNAs . To deteLsd1 mRNA, a target of the R783H Trim71 . Wn. Wn21]. Ctnnb1 mRNA contributes to the differentiation defects of R595H-Trim71 mESCs, we deleted the R595H-Trim71 binding site in the 3′ UTR of Ctnnb1 mRNA  , which mCtnnb1 mRNA, leading to attenuated Wnt/β-catenin signaling. These results strongly suggest that dysregulation of the Wnt/β-catenin signaling pathway at the posttranscriptional level is important for the pathogenesis of CH mediated by the R595H mutation in Trim71. Moreover, our findings indicate the two CH-associated mutations are gain-of-function mutations, consistent with monoallelic mutations in Trim71 causing CH in human.We discovered that although two different CH-associated mutations in Trim71 lead to similar differentiation defects in mESCs, they do so via distinct mechanisms, namely, the two Trim71 mutants ectopically bind to and translationally repress different mRNAs. We determined that R595H-Trim71 disrupts mESC differentiation because it specifically represses the translation of R595H/R595H, and Trim71R595H/+ mESCs were less proliferative than WT-mESCs upon N2B27-induced neural differentiation [Recent studies in mice showed that CH-associated mutations result in prenatal hydrocephalus in mice , indicatntiation .Our findings complement this study . InsteadR595H/+ mice and mice with neuroprogenitor-specific KO of Trim71 survive until birth, with approximately 16% to 25% displaying ventriculomegaly and progressive macrocephaly [R595H/R595H and Trim71-KO mice are embryonic lethal and displayed defects in neural tube formation [−/+ mice are viable, fertile, and lack abnormalities [Monoallelic mutations in Trim71 result in CH in human , suggestocephaly , whereasormation . In addimalities , suggestmalities , argue tOur studies and others showed that R595H-Trim71 and R783H-Trim71 have reduced binding to many WT-Trim71 target mRNAs. However, our results indicate that each of the two CH-associated Trim71 mutants also bind a new set of target mRNAs. Consistent with this finding, the RNA binding results from the recent study  indicateTrim71 interacts with RNA targets through the NHL domain . The resCtnnb1 mRNA and reducing the levels of β-catenin, resulting in stem cell differentiation defects. Intriguingly, it was recently shown that deletion of β-catenin in neural stem/progenitor cells also leads to CH in mice [Ralb has decreased β-catenin association in R595H-Trim71 mutant mESCs and that reduced expression of Ralb also leads to differentiation defects. We anticipate that uncovering additional Wnt/β-catenin target genes that are dysregulated in R595H-Trim71 mESCs will reveal new insights into CH pathogenesis.The Wnt/β-catenin signaling pathway plays important roles in diverse developmental processes . Our res in mice . These o in mice , therefoAll the antibodies, oligonucleotides, and plasmids used in this study are listed in All the mESCs were derived from the ES-E14TG2a line (ATCC CRL-1821) and were cultured in either 15% FBS + Lif or 2i + Lif medium on 0.5% gelatin-coated plates at 37°C with 5% CO2.sgRNA-expressing vector and donor oligo/plasmid were cotransfected into mESCs using Fugene6. Transfected cells were single-cell sorted to 96-well plates, 48 hours posttransfection. The desired clones were selected based on the PCR genotyping, Sanger sequencing, and western blot analysis.Total proteins were extracted by RIPA buffer, quantified using BCA assay kit, and then resolved by SDS-PAGE gels. Western blotting was performed using a BlotCycler (Precision Biosystems) with the indicated antibodies. The signals on the membranes were generated with the Western ECL substrate (Bio-Rad) and imaged with an ImageQuant LAS 500 instrument .RNA was isolated using Trizol with DNase1 treatment and then reverse transcribed into cDNA using random hexamers and Superscript2 reverse transcriptase. Quantitative real-time PCR was performed in triplicate for each sample by CFX96 real-time PCR detection system (Bio-Rad) with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad).The colony formation assay and the exit pluripotency assay were performed as described previously . For theFor immunofluorescence staining, cells cultured on chamber sliders were fixed with 4% PFA for 20 minutes at room temperature and then permeabilized with 0.2% Triton X-100 in PBS. Cells were blocked with 3% BSA in PBS and subjected to staining with the indicated antibodies diluted in blocking solution supplemented with 0.1% Tween-20 for 2 hours at room temperature. Cells were then washed and stained with Hoechst Staining Dye for 10 minutes and then mounted with mounting medium. The images were acquired by a Nikon Eclipse fluorescence microscope and analyzed by ImageJ.The luciferase assays were performed using the Dual-Glo Luciferase Assay System (Promega) on a GloMax 20/20 Luminometer (Promega).Polysome analysis was performed using the methods described previously .CLIP-seq and peaking calling was performed using the methods described previously ,31. PolyS1 FigA) The locations of the two CH-associated in Trim71. (B) Outline of the genome editing procedure. (C) Work flow of generating the two CH-associated mutations in mESCs. (D) Sanger sequencing results verifying the R595H mutation in mESCs. (E) Sanger sequencing results verifying the R783H mutation in mESCs. CH, congenital hydrocephalus; mESC, mouse embryonic stem cell.((EPS)Click here for additional data file.S2 Figp > 0.05). The data are available in The mESCs were cultured in 15% FBS + Lif for 7 days, and the resultant colonies were fixed and stained for AP. The colony morphology and AP intensity were evaluated through microscopy. To determine the percentage of undifferentiated colonies, 100–200 colonies were examined each time. The results represent the means (± SD) of three independent experiments. One-way ANOVA was used to determine the significance of the difference, n.s. not significant ((EPS)Click here for additional data file.S3 FigA) Proliferation of mESCs under the stemness condition. The cells were cultured in the 2i + Lif medium, and the cell proliferation was monitored by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). (B) Proliferation of mESCs under the differentiating condition. The cells were cultured in the 15% FBS—Lif medium, and the cell proliferation was monitored by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). (C) Apoptosis of mESCs under the stemness condition. The cells were cultured in the 2i + Lif medium, and the cellular apoptotic state was monitored by annexin-V and PI staining followed by flow cytometry analysis. (D) Apoptosis of mESCs under the differentiating condition. The cells were cultured in the 2i + Lif medium, and the cellular apoptotic state was monitored by annexin-V and PI staining followed by flow cytometry analysis. The results represent the means (± SD) of three independent experiments. *p < 0.05, n.s. not significant (p > 0.05) by one-way ANOVA. The data for A-D are available in ((EPS)Click here for additional data file.S4 Figp < 0.05, n.s. not significant (p > 0.05) by one-way ANOVA. The data are available in 18S rRNA was used for the normalization in the gene quantification by qRT-PCR. The qRT-PCR results represent the means (± SD) of three independent experiments. *(EPS)Click here for additional data file.S5 FigA) Work flow of the RNA-seq analysis. (B) Differentially expressed genes in the WT and R595H mESCs. (C) Expression of the target RNAs of the R595H Trim71 mutant in the WT and the R595H mESCs. The differentially expressed genes were identified by the edgeR package. The expression level of each gene is the average of the three biological replicates. CPM, counts per million reads; mESC, mouse embryonic stem cell; WT, wild-type.((EPS)Click here for additional data file.S6 Figp < 0.05, n.s. not significant (p > 0.05) by one-way ANOVA. CH, congenital hydrocephalus; qRT-PCR, quantitative real-time PCR.A representative western blotting the NCCIT cells expressing an empty vector, HA-hTRIM71, HA-hTRIM71(R608H), and HA-hTrim71(R796H). GAPDH was used for normalization in the quantification of Lsd1 protein levels, and 18S rRNA was used for normalization in the qRT-PCR quantification of Lsd1 mRNA levels. The quantification results represent the means (± SD) of three independent experiments. *(EPS)Click here for additional data file.S7 Figp > 0.05). The data are available in The exit pluripotency assay was performed on the indicated mESCs, and the resultant colonies were fixed and stained for AP. The colony morphology and AP intensity were evaluated through microscopy. To determine the percentage of undifferentiated colonies, 100–200 colonies were examined each time. The results represent the means (± SD) of three independent experiments. One-way ANOVA was used to determine the significance of the difference, n.s. not significant ((EPS)Click here for additional data file.S8 FigA) Distribution of the Ctnnb1-associated genomic regions in the mouse genome. Only the genomic regions identified in two independent Cut&Run assays were retained for the analysis. (B) Cut&Run and ChIP-seq data at the Sp5 locus, a classical target gene of the Wnt/beta-Catenin signaling. The ChIP-seq data were from GSE131119 of Gene Expression Omnibus. The data for A are available in ((EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(CSV)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S7 Data(XLSX)Click here for additional data file.S8 Data(XLSX)Click here for additional data file.S9 Data(XLSX)Click here for additional data file.S10 Data(XLSX)Click here for additional data file.S11 Data(XLSX)Click here for additional data file.S12 Data(XLSX)Click here for additional data file.


Patients with relapsed and refractory T-cell malignancies have poor outlook and limited treatment options. Recently, adoptive cell therapy has emerged as a promising therapy for patients with T-cell malignancies. In this review, we examine the current progress on adoptive cell therapy for T-cell malignancies and discuss the potential future directions.T-cell malignancies are often aggressive and associated with poor prognoses. Adoptive cell therapy has recently shown promise as a new line of therapy for patients with hematological malignancies. However, there are currently challenges in applying adoptive cell therapy to T-cell malignancies. Various approaches have been examined in preclinical and clinical studies to overcome these obstacles. This review aims to provide an overview of the recent progress on adoptive cell therapy for T-cell malignancies. The benefits and drawbacks of different types of adoptive cell therapy are discussed. The potential advantages and current applications of innate immune cell-based adoptive cell therapy for T cell malignancies are emphasized. T-cell malignancies are a heterogenous group of hematological cancers originating from dysfunctional precursor and mature T cells. T-cell malignancies can be broadly categorized into T-cell leukemias and T-cell lymphomas, with various subtypes based on the developmental stage and the location of T cells. T-cell malignancies are often aggressive and associated with poor prognoses. Among T-cell malignancies, more aggressive forms include T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL). T-ALL is a neoplasm of lymphoblasts committed to the T cell lineage and represents 15 to 25% of pediatric and adult ALL. PTCL affects mature T cells and accounts for 10 to 15% of non-Hodgkin’s lymphoma (NHL). The current first-line treatment for T-ALL and PTCL involves intensive multiagent combination chemotherapy. However, the overall survival of adult patients with T-ALL is less than 50% due to treatment-related toxicities and disease relapse ,2, whileImmunotherapy has been coined as the “fourth pillar” of cancer treatment, an addition to the traditional three pillars of surgery, radiation, and chemotherapy. Immunotherapy is an emerging branch of medicine that modifies the host immune system or utilizes components of the immune system to treat cancer. In particular, adoptive cell therapy (ACT) has gained interest as an immunotherapy and achieved promising clinical results for hematological cancers, particularly for B-cell malignancies. ACT involves the infusion of ex vivo expanded immune cells into patients for treating cancers, ranging from re-infusing immune cells taken from specific tumor sites to infusing genetically modified donor-derived immune cells. Currently, four major types of cells are used in ACT: tumor-infiltrating lymphocytes (TILs), transgenic T cell receptor (tgTCR)-modified T cells (tgTCR-T cells), chimeric antigen receptor (CAR)-modified T cells (CAR-T cells), and unmodified or modified innate immune cells . One form of ACT involves the use of TILs. TILs comprise intratumor lymphocytes that can recognize and attack tumor cells. The procedure of TIL-based ACT was first developed for melanoma patients in the 1980s . In TIL-The second type of ACT involves tgTCRs to improve tumor recognition and the function of lymphocytes. Early studies showed that antigen specificity of T cells could be modified via tgTCR gene transfer . T cellsA more popular approach of ACT utilizes CAR-T cells. CARs are artificial receptors that are generated by combining the signaling domain of a TCR with a single chain variable fragment (scFv) specific for a target antigen. Unlike TCRs, CARs can redirect the cytotoxicity of T cells against target antigens independent of MHC, albeit generally against surface-expressed antigens. In contrast with TIL, CAR-T cell therapy has primarily been evaluated for use in hematological cancers. Notably, CAR-T cell therapy has achieved significant clinical success against B-cell malignancies, and the first CAR-T cell therapy was approved in 2017 for CD19-directed CAR-T cell therapy against r/r B-ALL and diffuse large B cell lymphoma (DLBCL). In a clinical study of CD19 CAR-T cell therapy for DLBCL, 58% of patients showed complete response, and 25% of patients showed partial response , highlig+ and CD8+ T lymphocytes for treating cancers, studies have also recently gained interest in developing ACT using other immune cell types. Innate immune cells, such as natural killer (NK) cells, gamma delta (γδ) T cells, and double negative (DN) T cells, are currently being investigated for use in ACT against several hematological malignancies, including AML, B-cell malignancies, and T-cell malignancies. Each of these immune cells has shown unique advantages, limitations, and suitability for ACT due to their distinct characteristics and functions in the immune system. While the use of innate immune cell-based ACT for T-cell malignancies is still in its infancy, there have been some encouraging results.While ACT conventionally utilizes CD4The success of CAR-T cell therapy for B-cell malignancies has paved the way for the development of CAR-T cell therapy for T-cell malignancies. The potential of using CAR-T cells to target T-cell malignancies have been demonstrated in a number of preclinical studies, which have been extensively reviewed recently ,18,19,20The fratricide or self-killing of CAR-T cells is a major obstacle encountered for the development of CAR-T cell therapy for T-cell malignancies. The use of CAR-T cell therapy necessitates the selection of targetable antigens present on tumor cells. Ideally, these antigens should be lineage-restricted and commonly found on tumors, but not expressed on hematopoietic stem cells to reduce toxic side effects. B-lineage-specific antigens, such as CD19, CD20, and CD22, are typically selected as targets for CARs to treat B-cell malignancies. For T-cell malignancies, potentially targetable antigens include pan T-lineage antigens, such as CD3, CD4, CD5 and CD7. These antigens are highly expressed in T-cell malignancies  but are − blasts, only CD3+ cells were selected for CAR-T cell production [−CD8− T cells, only CD4+ and CD8+ T cells were used [The second challenge lies in the potential contamination of autologous CAR-T cell products. All FDA-approved CAR-T cell therapy uses autologous T cell from the peripheral blood of patients. In a case study, it was observed that the transduction of a single leukemic B cell with anti-CD19-CAR led to the masking of CD19 antigen on the leukemic cell and subsequent antigen-positive disease relapse . In patioduction , while fere used . The finere used ,23. NoneT-cell aplasia is the third obstacle. T cells play an important role in the immune system and are essential in fighting off infections and cancer development. The use of CAR-T cells that target T-lineage antigens would eliminate both malignant and normal host T cells, which may result in T-cell aplasia. While B-cell aplasia is also seen from CD19-CAR-T cell therapy, it is well tolerated in the clinic after immunoglobulin replacement treatment. In contrast, the effect of T-cell aplasia is not well studied and may result in AIDS-like immunodeficiency. Currently, there is no effective treatment for T-cell aplasia.Recently, studies have employed different strategies to overcome these challenges. These strategies predominantly involve additional genetic modifications of CAR-T cells but can also include other methods. The currently published preclinical approaches are summarized in − or CD7− specific CAR-T cells, studies have used gene editing tools such as TALEN and CRISPR/Cas9 [To avoid the fratricide of CAR-T cells, the best strategy is to select target antigens expressed only on tumors or shared with restricted T cell subsets. For example, CD37 is absent on T cells but found in some cases of PTCL , which mSPR/Cas9 ,30 or a SPR/Cas9  to abrogSPR/Cas9 . In thisSPR/Cas9 . Unedite+ alloreactive T cells. Subsequently, patients are infused with allogeneic CAR-T cell products that are knocked out of CD52, which allows the CAR-T cells to be resistant to anti-CD52 antibody treatment [As previously mentioned, using allogeneic T cells is a strategy to avoid autologous CAR-T cell product contamination. This approach necessitates further modifications to allogeneic T cells to overcome the issues of GvHD and allorejection. Acute GvHD is commonly mediated by alloreactive donor TCRs. To avoid GvHD, studies have knocked out the TCRα chain and TCRαβ from T cells using CRISPR/Cas9 and TALEN-mediated gene editing . These sreatment . Yet, th+ T-cell malignancies while sparing TRBC2+ T cells [Unlike B-cell aplasia, T-cell aplasia can lead to life-threatening consequences. One strategy to mitigate T-cell aplasia is to target antigens expressed only on specific T-cell subsets to avoid completely eradicating T cells. For example, since TRBC1 and TRBC2 expression on T cells are mutually exclusive, a study has demonstrated that TRBC1-specific CAR-T cells can target TRBC1 T cells . However T cells . To limi T cells . Since t T cells . HoweverOverall, the preclinical studies have demonstrated the feasibility of genetically modifying CAR-T cells and showed the requirement for additional processes to mitigate challenges associated with targeting T-cell malignancies.To date, there are at least four completed phase I clinical trials, two terminated phase I clinical trials, and 53 ongoing phase I and phase II clinical trials for T-cell malignancies using CAR-T cells, 35 of which are at the recruitment stage. These 59 studies focus on the use of CD4 (5 studies), CD5 (5 studies), CD7 (28 studies), CD30 (17 studies), CD37 (1 study), CD123 (1 study), CD147 (1 study), or TRBC1 (2 studies)-specific CARs to target a diverse range of T-cell malignancies, including T-ALL, PTCL, T cell lymphoblastic lymphoma (T-LBL), anaplastic large cell lymphoma (ALCL), adult T cell leukemia/lymphoma, angioimmunoblastic T cell lymphoma (AITL), enteropathy-associated T cell lymphoma, and extramedullary NK/T cell lymphoma. These clinical trials primarily investigate autologous or HSCT-donor-derived CAR-T cells , healthy donor-derived allogeneic CAR-T cells , or both  for patients with r/r disease after chemotherapy with or without prior HSCT. A few trials evaluate HSCT-donor derived CAR-T cell therapy for patients post-HSCT to prevent disease relapse. Among CD7-specific CAR-T cells, various techniques are utilized to prevent fratricide, including CD7 expression blockade and genetic abrogation. For most studies on allogeneic donor-derived therapy, “universal CAR-T cells” are manufactured with TCRα subunit constant (TRAC) disruption to avoid GvHD, while virus-specific T cells are used in one study to navigate the issue of GvHD. Aside from active clinical trials, there is also a follow-up study examining the long-term effects of CD7-specific CAR (NCT05509855). All completed and ongoing clinical trials on CAR-T cell therapy for T-cell malignancies are listed in −, CD5−, CD7, and TRBC1-specific CAR-T cells for r/r T cell leukemias and lymphomas and CD30-specific CAR-T cells for r/r T cell lymphomas. Most trials have achieved impressive results at one month follow-up. Of 13 trials where more than two patients (n = 3 to n = 22) were enrolled, 12 trials have reported complete remission (CR) rates that range from 66 to 100%, whereas in one trial the overall response rate (ORR) was 36.6% although 57% patients remained in stable disease (SD). However, trials with longer-term follow-ups showed that the responses achieved were not durable. For example, in one trial (NCT04572308), patients were treated with “naturally selected” CD7-specific CAR (NS7CAR) T cells [Sixteen of the completed and ongoing phase I clinical trials have published full manuscripts (3 studies) or preliminary results (13 studies), which are summarized in  T cells . FollowiDespite the potential therapeutic efficacy in treating T-cell malignancies, these clinical trials also highlighted some limitations and risks involved in using CAR-T cell therapy. In all studies, patients experienced mild to moderate, and sometimes severe, adverse events. Of note, all patients in the two aforementioned studies experienced adverse events, where 19 out of 20 and 7 out of 8 patients experienced cytokine release syndrome (CRS) and two had neurotoxicities ,23. In oFurthermore, there are additional issues that may be involved in using CAR-T cell therapy for T-cell malignancies due to the need to avoid fratricide, product contamination with blasts and T-cell aplasia. For example, with the use of gene editing tools, there are risks of unwanted on-target and off-target effects, including pan-cytopenia and tumorigenesis. Such risks are only elevated with the increased complexity of gene modifications. Moreover, multiple gene modifications render the manufacturing process of CAR-T cells more complicated and costly. It is difficult, time-consuming, and laborious to generate and isolate T cells that have undergone multiple modifications with 100% or high enough purity. Preclinical studies have reported a poor expansion and yield for CAR-T cells following the knockout of CD3 and TCRαβ  and foun+ and CD8+ T-lymphocytes for T-cell malignancies, other immune cells are being investigated for use in ACT. The cell types primarily encompass innate immune cells, such as NK cells, γδ T cells, and DNTs. These immune cells occupy unique roles in the immune system and have distinct characteristics that can be leveraged to develop off-the-shelf allogeneic ACT. These innate immune cells are currently being studied as standalone and CAR-based therapies. The potential advantages and limitations of ACT using these immune cell types are summarized in Due to the unsolved issues posed by using conventional CD4NK cells are innate immune cells defined by the expression of CD56 without CD3 or TCR. NK cells provide a first line of defense against malignant or virally infected cells. Unlike T cells, NK cells recognize malignant cells independent of antigen presentation and MHC restriction. Instead, NK cell activity is regulated by signals from an array of activating, inhibitory, and cytokine receptors that interact with specific ligands. Some important activating receptors include NK group 2D (NKG2D) and nature cytotoxicity receptors (NCRs), such as NK protein 30 (NKp30) and NK protein 44 (NKp44). These activating receptors bind to stress-induced molecules often expressed on malignant cells. To prevent self-recognition, NK cells express inhibitory receptors, such as killer cell immunoglobulin-like receptors (KIRs), which send inhibitory signals upon interactions with healthy self MHC-class I molecules. While tumors can often evade T cell recognition by downregulating MHC molecules, NK cells become activated when encountering cells that do not express self MHC molecules. Upon activation, NK cells produce rapid effector functions, releasing proinflammatory cytokines and cytotoxic granules to promote immune response and cause tumor cell lysis. Further, NK cells express CD16a, which can be activated by binding to antibodies deposited on malignant cells and induce antibody-dependent cell-mediated cytotoxicity (ADCC).The antitumor effect of allogeneic NK cells was first demonstrated in HSCT for hematological malignancies. In T cell-depleted allogeneic HSCT, donor NK cells were the first immune cell population to recover and control residual malignant cells ,72,73. IFor T-cell malignancies, NK cell-based ACT have only been in evaluation for concurrent use with chemotherapy or post-HSCT. To date, there have been 5 clinical trials that investigated NK cell infusion for patients with r/r T-cell leukemia and lymphoma. Two of the 5 studies have been completed but have yet to report the outcomes. For the remaining 3 studies, one is in the recruitment stage, one has unknown status, and the other has been terminated due to low recruitment. Several trials have also investigated NK cell infusion post-HSCT for hematological malignancies but have not specified if any patients with T-cell malignancies are enrolled. The scarcity of clinical trials for T-cell malignancies may be related to early findings that donor NK cells in KIR-mismatched HSCT were beneficial for pediatric ALL but not adult ALL, which lacks adhesion molecules essential for NK cell activation. The clinical trials using NK cell therapy for T-cell malignancies are summarized in +, CD4+, CD5+ and CD7+ T-cell malignancies. CAR-NK cells have shown potent antitumor activity against T-ALL and PTCL in vitro and in xenograft models [To utilize and improve the endogenous cytotoxicity of NK cells against tumors, studies have armoured NK cells with CARs. CAR-modified NK cell therapy is an emerging area of study, particularly in T-cell malignancies as NK cells do not share T-lineage antigens and are safe to be used in allogeneic settings. Since T and NK cells differ in hematopoietic lineage, fratricide can be avoided without modifying the surface antigen expression on NK cells. With these advantages, preclinical studies have used CAR-NK cells to target CD3t models ,81,82,83t models ,85. In ot models . FurtherHowever, there are also several challenges involved in using NK cells. NK cell expansion and persistence in recipients depend on cytokines such as IL-2 or IL-15, which are associated with toxicities when given systemically. In allogeneic NK cell therapy, there is a risk of immune rejection by host cells, especially in cases where systemic IL-15 supplementation is used to promote NK cell expansion . StudiesWith the advances in induced pluripotent stem cells (iPSC), iPSC-derived NK cells have also been investigated as a potential source of NK cells . iPSCs hBesides the issues encountered by NK cells, CAR-NK cells face additional challenges during the manufacturing process. NK cells have a lower transduction rate than conventional T cells, which may be due to the intrinsic resistance of NK cells against viral transduction . Since C−CD8−, and 30% are CD4+ or CD8+ [γδ T cells are a small subset of T lymphocytes that express a combination of a Vγ TCR chain paired with a Vδ chain. γδ T cells account for 1–10% of circulating T cells in the peripheral blood and more than 20% of intraepithelial T cells in the intestine ,112. Aro or CD8+ . γδ T ce or CD8+ ,115. Unl or CD8+ ,117. γδ  or CD8+ ,119. Of  or CD8+ ,122,123. or CD8+ . γδ T ce or CD8+ ,126, whi or CD8+ ,128, whi or CD8+ , which a or CD8+ . In addi or CD8+ ,132. In preclinical studies, γδ T cells have been shown to have antitumor effects against a broad spectrum of hematological and solid malignancies, including acute myeloid leukemia (AML) , B cell + T-cell malignancies without fratricide. αβ TCR is expressed on >95% of PTCL [+ B-cell malignancies and NKG2D+ solid tumors. In one trial of allogeneic off-the-shelf CD20-CAR γδ T cells for r/r B-cell lymphoma (NCT04735471) [To improve the efficacy of γδ T cells, researchers have developed CAR γδ T cells. Studies have shown that γδ T cells can achieve similar levels of transduction efficiency as αβ T cells. In preclinical studies, CAR γδ T cells have been shown to be effective against various malignancies, including B-cell malignancies ,150, neu of PTCL  and >30% of PTCL . A study of PTCL , suggest of PTCL , which m4735471) , 11 out + T-ALL in vitro. This suggests that surface antigen modifications of γδ T cells are likely necessary to avoid fratricide while preserving higher antitumor efficacy. Nonetheless, there are currently several limitations to using γδ T cell therapy for T-cell malignancies. Similar to NK cells, the short lifespan of γδ T cells is both an advantage for avoiding T-cell aplasia and a disadvantage for lacking persistence. Due to the short lifespan of γδ T cells , multipl+ T cells and TCRγδ+ T cells, with proportions that vary between individuals. The origin of DNTs is currently not well understood. There are evidence supporting that peripheral DNTs originate from both thymic and non-thymic origin as thymectomized mice showed reduced, but detectable, levels of DNTs [+ or TCRγδ+ DNTs, which possibly leave the thymus and expand in the periphery [+ or CD8+ T cells can give rise to peripheral DNTs [+ DNTs and TCRγδ+ DNTs have been shown to have effector and regulatory functions and are capable of recognizing target antigens without MHC restriction [+ DNTs and TCRγδ+ DNTs [DNTs are mature unconventional T cells defined by the expression of CD3 without CD4 or CD8 expression. Circulating DNTs comprise 3–5% of peripheral blood T lymphocytes, while tissue-resident DNTs have been observed in various organs, including the intestine, kidney, lung, liver, heart, genital tract, and central nervous system ,164,165. of DNTs . In the eriphery ,168. Thyral DNTs ,171,172.triction ,174. DNTtriction ,176. A sγδ+ DNTs , suggest+ T cells [Of interest, our lab has shown that ex vivo expanded DNTs have antitumor effects against a variety of cancer types, including AML and non-small cell lung cancer ,179. DNT T cells . Ex vivo T cells , highlig T cells . DNTs sh T cells . Further T cells ,183, and T cells . These c T cells . This stIn addition, it was shown that the antitumor efficacy of DNTs can be improved by CAR transduction. In a recent report, CD19-CAR DNTs showed potent efficacy against B-cell malignancies, comparable to that of CD19-CAR conventional T cells while retaining its off-the-shelf characteristics . The stuNonetheless, DNTs constitute a small percentage of the peripheral blood, and there is donor variability in the number DNTs that can be expanded. We have studied DNTs from about 200 healthy donors and observed that DNTs from majority of donors can be easily expanded ex vivo and possess potent antitumor activity. However, about 15% healthy donors’ DNTs show poor expansion rate and low cytotoxicity against tumor cell lines. Therefore, pre-screening of donors to exclude 15% of poor DNT-expanders is needed. Murine and human studies show that αβ-DNTs have potent immune-regulatory functions, where it can prevent GvHD and graft rejections ,193,194.In addition to conventional T cells and innate immune cells, other emerging cell types may be suitable for use in ACT, such as NK T cells  and macrHowever, several challenges remain in using ACT for T-cell malignancies and other malignancies in general. There are currently two conflicting needs for T-cell malignancies: the need for T cell persistence to achieve long-term therapeutic efficacy and the need to avoid T-cell aplasia. It is thus paramount to find new tumor-restricted or subset-restricted targets to minimize the effect of T-cell aplasia. For the use of ACT, suppressive tumor microenvironments, antigen escape, and lack of cellular infiltration continue to be significant obstacles. As mentioned above, identification of antigens expressed on T-cell malignancies but not on normal T cells, appropriate uses of ACT as bridge therapy and suicide genes need to be further investigated. Further, studies are now steering towards using ACT in combination with other forms of immunotherapy, such as checkpoint inhibitors, oncolytic viruses , the stiACT is emerging as a new line of therapy for patients with hematological malignancies. While there are currently challenges in applying ACT to T-cell malignancies, preclinical and clinical studies investigate various approaches to navigate these challenges. Although current ACT typically uses the autologous conventional T cells, different ACT types are being developed to harness the unique functions and properties of other immune cell types for allogeneic therapy. Innate immune cell-based therapies have demonstrated ample potential and garnered interest as a promising area of research for T-cell malignancies.