The efficacy of therapeutic T-cells is limited by a lack of positive signals and excess inhibitory signaling in tumor microenvironments. We previously showed that a constitutively active IL7 receptor (C7R) enhanced the persistence, expansion, and anti-tumor activity of T-cells expressing chimeric antigen receptors (CARs), and C7R-modified GD2.CAR T-cells are currently undergoing clinical trials. To determine if the C7R could also enhance the activity of T-cells recognizing tumors via their native T-cell receptors (TCRs), we evaluated its effects in Epstein–Barr virus (EBV)-specific T-cells (EBVSTs) that have produced clinical benefits in patients with EBV-associated malignancies. EBVSTs were generated by stimulation of peripheral blood T-cells with overlapping peptide libraries spanning the EBV lymphoma antigens, LMP1, LMP2, and EBNA 1, followed by retroviral vector transduction to express the C7R. The C7R increased STAT5 signaling in EBVSTs and enhanced their expansion over 30 days of culture in the presence or absence of exogenous cytokines. C7R-EBVSTs maintained EBV antigen specificity but were dependent on TCR stimulation for continued expansion. C7R-EBVSTs produced more rapid lymphoma control in a murine xenograft model than unmodified EBVSTs and persisted for longer. The findings have led to a clinical trial, evaluating C7R-EBVSTs for the treatment of refractory or relapsed EBV-positive lymphoma (NCT04664179). Epstein–Barr Virus (EBV) is linked to a range of malignancies including about 30% of Hodgkin and non-Hodgkin lymphomas in which four viral latent cycle proteins, EBNA-1, LMP1, LMP2, and BARF1 (Type 2 latency antigens (T2-Ags)) are expressed ,5, theirThe expansion and survival of adoptively transferred T-cells is limited by the suppressive tumor-microenvironment (TME) ,11,12 thThe early success of high-dose IL-2 cytokine administration along with tumor-infiltrating lymphocytes (TILs) ,18 estabTo enhance the proliferation, survival, and persistence of adoptively transferred EBVSTs, we have focused on providing IL-7 cytokine signaling. To circumvent the toxicity and limitations associated with secreted cytokines, we used a constitutively active IL-7 receptor (IL7R). Cysteine and/or proline insertions in the transmembrane domain of the IL7R α chain produces homodimerization resulting in phosphorylation of JAK1 and activating STAT5 signaling ,32. To eThe C7R comprises a CD34-derived ectodomain, linked to the transmembrane and intracellular domains of the IL7Rα chain, carrying in the transmembrane domain that results in homodimerization as outlined in p = 0.047]) over 16 days (5 cells that produce IFNγ in response to antigen stimulation (51Cr chromium release assays (p = 0.031) (p = 0.031) (CM), or effector memory (TEM) (CD45RO+ CCR7-) CD3+ T-cells  D, which = 0.031) E indicat= 0.031) F, or in  T-cells G, allayip = 0.044, CM-cells in C7R-EBVSTs cultured both in the presence or absence of cytokines compared to NT-EBVSTs, although this was not statistically significant  expressing NT- or C7R-EBVSTs, and measured tumor volume and T-cell bioluminescence to evaluate anti-tumor efficacy and T-cells’ persistence over time. Tumor clearance, as determined by a lack of palpable tumor, was observed on day 67 after C7R-EBVST infusion. At this time, tumors measured 239.49 mm3 ± 61.96 mm3 in mice receiving NT-EBVSTs (p = 0.004)  subcutaneously in NSG mice. Ten days after implantation, when tumors were palpable, we adoptively transferred 2.5 × 10T-EBVSTs A. The suT-EBVSTs B,C. The = 0.004) C corresp= 0.004) B,C indic= 0.004) D.The lymphoma TME is not only immunosuppressive but lacks immunostimulatory cytokines critical to EBVST survival and persistence . The conWhile C7R enhanced EBVST proliferation, it did not produce uncontrolled T-cell outgrowth either in vitro or in our in vivo models in line with the lack of autonomous growth reported by Shum et al., 2017 , who shoCM in C7R-EBVSTs cultured in the presence or absence of cytokines compared to NT-EBVSTs after long-term in-vitro culture, suggesting that C7R supports and maintains TCM subsets.IL-7 cytokine-driven STAT5 activity is negatively regulated by the suppression of cytokine signaling-1 (SOCS-1) and/or dephosphorylation by SHP2 , therefoSTAT5-mediated signaling in T-cells provides resistance to immunosuppressive agents such as M2 macrophages, PD-1-PD-L1 interaction, and TGF-β within the tumor microenvironment ,40. ShumIn summary, we have demonstrated the feasibility of expressing C7R in EBVSTs and its ability to enhance the persistence and potency of EBVSTs targeting EBV-associated lymphoma. Though our study is limited by our murine model in assessing the safety profile of C7R-EBSVTs, the lack of autonomous outgrowth of C7R-expressing T-cells in both CAR T- and EBVSTs supports the clinical translation. Nor could our immunodeficient murine model determine the ability of the C7R to counteract a fully functional TME. If successful in improving the clinical potency of EBVSTs for lymphoma, the C7R could be applied to EBVSTs targeting more intractable EBV-associated malignancies, such as nasopharyngeal carcinoma (NPC) and gastric adenocarcinoma (GC), as well as other oncogenic viruses such human papillomavirus (HPV). Future directions will investigate whether C7R provides resistance to specific immunosuppressive components of the TME such as ROS, IDO, NOS, suppressive cytokines , or inhibitory ligands . C7R-EBVSTs are currently under clinical and immunological evaluation in patients with EBV+ lymphoma NCT04664179) [64179 [6]Blood samples were collected with informed consent from healthy EBV seropositive individuals based on Baylor College of Medicine Institutional Review Board (IRB)-approved protocols. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep gradients  and used for the generation of virus-specific T cells (VSTs) and activated T cells (ATCs). HLA typing was performed by the Houston Methodist HLA Laboratory.PBMCs were stimulated using CD3  and CD28 antibodies  in the presence of interleukin-2 (IL-2)  at 50 units per mL following established procedures as previously documented and consistent with standard laboratory practices ,34. PrioOverlapping peptide libraries (15 mer peptides overlapping by 11 amino acids) spanning the complete protein sequences of T2-Ags  were purchased from JPT technologies , reconstituted at 200 micrograms/mL in DMSO , and stored at −80 °C. Pepmixes were thawed and reconstituted at the appropriate concentration as mentioned below for each experimental use.TM.ATCs and EBVSTs were cultured in VST medium , supplemented with 45% Click’s medium , 200 mM GlutaMAX TM-I , and 10% Fetal Bovine Serum . K562cs and LCLs were cultured in RPMI 1640 and enriched with 10% fetal bovine serum and 200 mM GlutamaxEBV-transformed B-lymphoblastoid cell lines (EBV-LCLs) were generated by infecting healthy donor PBMCs with concentrated virus from the B95-8 strain of EBV in the presence of 1 mg per mL of cyclosporin A, followed by culturing allowing the outgrowth of EBV-LCLs, as described previously ,42.The K562 costimulatory cell line (K562cs) was derived from a chronic erythroid leukemia cell line that lacks HLA class I and II molecules by lentiviral transduction with CD80, CD83, CD86, and 4-1BB ligand genes, and was generously provided by Dr. Carl June from the University of Pennsylvania, Perelman School of Medicine. In some experiments, the ULCL was used for co-stimulation in place of K562cs. The ULCL lacks HLA expression and was created within our research center using CRISPR-Cas9 gene-editing techniques.TTGTCCCAC insertion between base pairs 731 and 732 (IL7R*) was synthesized  and cloned in an SFG vector backbone. The IL7R* cDNA and the entire extracellular domain of CD34 (ΔCD34) are published in Shum et al. 2017  × 100.In-vitro cytolytic specificity of EBVSTs, as evaluated in 2. Bioluminescence measurements were taken with an IVIS Imaging system  following a 10 min wait period after the intraperitoneal administration of 150 mg/kg D-luciferin (Xenogen) per mouse. Total luminescence over the tumor area was visualized and quantified using Living Image software version 4.7.3 , with the region of interest (ROIs) encompassing the tumor region. All procedures were conducted in accordance with the Institutional Animal Care and Usage Committee (IACUC) approved protocol #AN5551 at Baylor College of Medicine.To evaluate the anti-tumor efficacy, and persistence of EBVSTs in-vivo, as described in t-test and/or as indicated in figure legends. Data are plotted as mean ± SEM unless otherwise indicated. Significance is denoted by p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) unless otherwise indicated.We used GraphPad Prism 7  for statistical analysis, using paired Students CMR, BO, and TS are co-authors of patent application WO2018038945A1, and the C7R technology is licensed to Gracell Biotechnologies .


Scientific Reports 10.1038/s41598-023-42171-5, published online 27 September 2023Correction to: The original version of this Article contained errors in Figure 12, where panels B and C as well as panels E and F were reversed. The original Figure The original Article has been corrected.


This decreased expression of PD-L1 by neonatal T-cells was also observed after stimulation with GBS, but not after stimulation with E. coli, the two most important pathogens in neonatal sepsis. Expression of the T-cell receptor CD3 and the co-stimulatory molecule CD28 did not differ between adult and neonatal T-cells upon bacterial stimulation. Decreased expression of ICM upon T-cell activation may be a reason for the increased risk of neonates to develop post-inflammatory diseases.Term and especially preterm neonates are much more susceptible to serious bacterial infections than adults. But not only the susceptibility to infection is increased in neonates, but also their risk for developing post-inflammatory diseases such as bronchopulmonary dysplasia (BPD) and periventricular leukomalacia (PVL). This may be due to an impaired ability to terminate inflammation. In the study presented here, we aimed to investigate the proliferative response and the expression of immune-checkpoint molecules (ICM) and activation markers on neonatal T-cells in comparison to adult T-cells with the hypothesis that an increased activation of neonatal T-cells may contribute to the failure of inflammation resolution observed in neonates. We show that neonatal CD4The online version contains supplementary material available at 10.1007/s12026-022-09340-6. Neonatal sepsis is one of the leading causes of infant death and, despite advances in the neonatal care, often results in poor outcome among survivors –3. The iCompared to adults, term and especially preterm neonates are much more vulnerable to serious bacterial infections , 9. InteIn adults, the host immune response to sepsis is traditionally considered to be characterized by an initial hyper-inflammatory phase, primarily driven by innate immune cells and a protracted anti-inflammatory phase leading to persistent immunosuppression and recurrent infections . This seICM are inhibitory receptors expressed by immune cells and mediating immunosuppressive signaling pathways in the context of T-cell activation. ICM play an important role in tolerance to auto-antigens and in the regulation of inflammation responses. In addition, certain pathogens, tumors, and leukemias use the expression of ICM to evade the T-cell response (immune escape) –18. In aThe immunosuppression induced by sepsis has not yet been defined in neonates . Actuall+ and CD8+ T-cells exhibit (1) an increased proliferative capacity and increased expression of activation markers in comparison to adult T-cells and (2) a decreased expression of ICM, especially PD-L1 on their surface. (3) The decreased expression of ICM by neonatal CD4+ T-cells was also observed after stimulation with Group B Streptococcus (GBS), one of the most important pathogens in neonatal sepsis. (4) Expression of the T-cell receptor CD3 and the co-stimulatory molecule CD28 did not differ between neonatal and adult T-cells upon bacterial stimulation.Here, we aimed to investigate the proliferative response and the activation state, as well as the expression of ICM on neonatal T-cells in comparison to adult T-cells with the hypothesis that decreased upregulation of ICM on neonatal T-cells upon bacterial stimulation may contribute to sustained inflammation. We show that upon stimulation with OKT3 neonatal CD4n = 32) immediately after cesarean section or vaginal delivery and placed in heparin-coated tubes. Collection of cord blood samples was anonymized; thus, no information on sex, exact gestational age, or mode of delivery was available. Children with intra-amniotic infection, defined by the German Society of Gynaecology and Obstetrics as increased maternal inflammatory markers without any other cause , fetal or maternal tachycardia, maternal fever (≥ 38.0 °C), painful uterus, and foul-smelling amniotic fluid, were excluded. Peripheral blood was collected from trained healthy adult volunteers .The study protocol was approved by the Ethics Committee of the Medical Faculty of Tübingen University (458/2019BO1). All mothers gave written informed consent prior to going into labor. Cord blood was collected from healthy term newborns  and separated by magnetic activated cell sorting (MACS) in an autoMACS® separator according to the manufacturer’s protocol (Miltenyi Biotec). Monocytes were separated from PBMC by two consecutive steps of panning (each 2 h) in cell culture flasks.Mononuclear cells from heparinized cord blood (CBMC) and peripheral blood (PBMC) were isolated by density gradient centrifugation . T-cells were separated from PBMC and CBMC after labeling with human CD46 cells/ml.Cells were then diluted in RPMI 1640 containing 10% heat-inactivated fetal calf serum , 1% penicillin/streptomycin (Biochrom), and 1% Glutamine (Biochrom) at a concentration of 2 × 106 cells per well) and stimulated with 0.01 µg/ml OKT3 .For stimulation of PBMC and CBMC with OKT3, cells were seeded to 24 well plates (2 × 106 cells/well) and stimulated with inactivated GBS in a multiplicity of infection (MOI) of 1:50.The culture of Group B streptococci was performed as previously described . BrieflyE. coli K1 was grown on agar plates. After 16 h, a single colony was picked and grown in Lennox L broth-medium  with or without supplements until early logarithmic growth. For stimulation of PBMC and CBMC, cells were seeded to 24 well plates (2 × 106 cells/ml) and stimulated with E. coli in a MOI of 1:50.A clinical isolate of E. coli.For inhibition of TRIF, the Pepinh-TRIF inhibitor or the appropriate control peptide  were added during stimulation with 5 cells in 100 μl RPMI media). Freshly isolated monocytes from a different adult donor were added as co-stimulatory cells in a 1:2 ratio. Cells were stimulated with different concentrations of OKT3 . After 96 h of incubation, cells were harvested and stained with anti-CD4-APC and anti-CD8-PE. CFSE fluorescence intensity was analyzed by flow cytometry. Data acquisition was performed with a FACSCalibur, and a FACSCantoII flow cytometer and data were analyzed via CellQuest and FlowJo (BD Biosciences).For analysis of T-cell proliferation of cord blood and adult T-cells, MACS-isolated T-cells were stained with carboxyfluorescein succinimidyl ester  according to the manufacturer’s instructions and seeded into round-bottom 96-well plates  controls to set thresholds for positive and negative stained cells. Gating strategy for CD4+ and CD8+ T-cells and strategy to set the threshold for ICM staining is depicted in Supplementary Fig. Antibodies used for extracellular staining of T-cells were purchased from BD Bioscience, Heidelberg, Germany , Miltenyi Biotec , and BioLegend, San Diego, USA . T-cell subpopulations were defined by staining with CD45RO and CCR7. Naïve T-cells were CD45ROt test. A p value of < 0.05 was considered statistically significant.Statistical analyses were done with GraphPad Prism version 9.1.2. Values were tested for Gaussian distribution using D’Agostino and Pearson omnibus normality test. Differences in proliferation assays and differences in the expression of surface ICM (percentages) were analyzed using the Wilcoxon matched-paired signed rank test. Differences in MFI were analyzed using the paired + T-cell proliferation 47.5% ± 18.9% (adult) vs. 69.5% ± 13.0% (cord blood), n = 6, p < 0.05, Fig. + T-cell proliferation 61.3% ± 25.2% (adult) vs. 81.8% ± 11.9% (cord blood), n = 6, p < 0.05, Fig. First, we asked whether there are differences in the proliferation of adult and neonatal T-cells upon stimulation of the T-cell receptor with OKT3. Therefore, we added monocytes isolated from adult PBMC either to T-cells isolated from CBMC or T-cells isolated from PBMC (different donor) and stimulated cells with OKT3 for 4 days. Here, we found a stronger proliferation in cord blood T-cells than in adult T-cells when both were cultured with adult monocytes . Upon OKT3-stimulation, about 70% of adult CD4+ T-cells expressed PD-1 and PD-L1, about 80% expressed PD-L2, and about 60% expressed CTLA-4. Overall, less CD8+ T-cells expressed ICM with about 20% of adult CD8+ T-cells expressing PD-1, 30% expressing PD-L1, 70% expressing PD-L2, and 60% expressing CTLA-4  but not in cord blood CD8+ T-cells , central memory (CD45RO+/CCR7+), and effector memory (CD45RO+/CCR7−) T-cell subsets. As expected, we found increased percentages of naïve T-cells and decreased percentages of effector memory CD4+ and CD8+ T-cells in cord blood samples compared to adult blood ) in adult and cord blood after OKT3 stimulation. Unstimulated CD4A-4 Fig. –P. Signills Fig. –P, and plls Fig. –D. SupplE. coli or GBS. Upon stimulation with both, E. coli and GBS about 40% of adult CD4+ T-cells expressed PD-1 and PD-L1, about 20% PD-L2 and only 10% CTLA-4  and the co-stimulatory molecule CD28 upon stimulation with lls Fig. –L. In coIt is well known that term and especially preterm infants are predisposed to bacterial infections such as sepsis. Moreover, the termination of inflammation seems to be disturbed leading to sustained inflammation , 26. Thi+ T-cells. (3) Also, upon stimulation with GBS, but not with E. coli, expression of ICM, especially that of PD-L1, was decreased on neonatal compared to adult CD4+ T-cells, while (4) expression of the T-cell receptor CD3 and the co-stimulatory molecule CD28 did not differ between neonatal and adult T-cells.In the present study, we found that in presence of adult monocytes (1) neonatal T-cells exhibited an increased proliferative capacity and an increased expression of T-cell activation markers in comparison to adult T-cells upon stimulation with OKT3. (2) This was accompanied by a decreased expression of ICM on neonatal T-cells, especially on CD4Our finding of increased proliferation of neonatal T-cells in comparison to adult T-cells is in line with previous reports also showing an increased proliferative response of cord blood T-cells stimulated by adult APC in comparison to adult T-cells –29, whilIn addition to the increased proliferation after OKT3 stimulation, we observed increased expression of activation markers CD25, CD38, CD69, and HLA-DR on neonatal compared to adult T-cells. In particular, a greater proportion of neonatal T-cells expressed CD25 and CD38. CD25 is part of the receptor for interleukin-2 (IL-2R) which is one of the cytokines inducing T-cell-proliferation, and increased expression of CD25 on neonatal T-cells may reflect increased susceptibility to IL-2-induced proliferation. The increased expression of CD38 on neonatal T-cells after OKT3 stimulation that accompanied their increased proliferative capacity in comparison to adult T-cells is in contrast to observations from Sandoval-Montes et al. who showed that T-cells expressing high levels of CD38 had a reduced proliferative capacity but an increased potential to produce pro-inflammatory cytokines . Despite+ T-cells showed a decreased expression of ICM compared to adult T-cells. This was confirmed, albeit to a lesser extent, after stimulation with GBS but not with E. coli. Co-stimulatory and co-inhibitory molecules elementarily determine the functional outcome of T-cell receptor signaling [After stimulation with OKT3, especially neonatal CD4ignaling . ICM canignaling , 40. A dignaling , 42. Theignaling . Correspignaling . To the ignaling . Data ofignaling ; however+ T-cells binding to PD-L1 induced STAT3-dependent “back signaling” that prevented T-cell activation and polarization [+ T-cells, induced their conversion into Tregs via the mitogen-activated protein kinase (MAPK) pathway [If the decreased expression of PD-L1 and to a lesser extent PD-L2 and CTLA-4 we observed on neonatal T-cells in comparison to adult T-cells after stimulation is the reason for their increased proliferative capacity remains unclear. While signaling via PD-1 in T-cells has been studied in detail (reviewed for example in ), the sirization . Recentl pathway , which c+ T-cells. This was particularly the case in memory T-cells [+ T-cells may represent cells with particularly high potential for adopting regulatory capabilities.In some samples, we observed two populations of PD-L1-expressing cells of which one expressed PD-L1 very high. We do not have a clear explanation for this. As mentioned before, Fanelli et al. recently showed that PD-L1 ligation induced a highly suppressive phenotype in CD4 T-cells . InteresE. coli. Recognition of GBS by host immune cells mainly involves toll like receptor 2 (TLR2) and myeloid differentiation factor 88 (MyD88) [E. coli is recognized by TLR4 and binding activates two alternative adaptor proteins, MyD88a and TIR domain-containing adapter-inducing interferon β (TRIF) [E. coli, which is transduced via both MyD88 and TRIF, which is well developed in neonates. However, we did not observe in our experiments that upregulation of PD-L1 expression in neonatal T-cells upon E. coli stimulation could be reversed by inhibition of TRIF  , while Eβ (TRIF) . For monβ (TRIF) , 54. In β (TRIF) . These dβ (TRIF) . If thes8 , while+ T-cells, decreased cell death, and enhanced the blood–brain barrier integrity [+ T-cells may contribute to the sustained inflammation of the lung and brain in neonates, but further investigations are needed.In vitro studies with adult cells showed a reduced lymphocyte apoptosis and recovery of effector immune cells after blocking PD-1 and PD-L1 in adult septic patients –60. In antegrity –63. ThesE. coli or GBS in neonates compared to adults, suggesting that TCR signaling and co-stimulation are not impaired in neonates. This is in line with previous studies also showing similar expression of CD3 and CD28 after stimulation with PMA in cord blood and adult T-cells [Lastly, we found no changes in the expression of T-cell receptor (TCR) and co-stimulatory molecule CD28 upon stimulation with  T-cells , 64, 65.E. coli. Decreased expression of ICM upon T-cell activation in neonatal cells may be a reason for the increased risk of term and preterm neonates to develop post-inflammatory diseases. More studies are needed to figure out if targeting immune checkpoint molecules in neonates may be a strategy to improve neonatal outcome.In conclusion, we show that neonatal T-cells have increased proliferative capacity upon stimulation with OKT3 in comparison to adult T-cells accompanied by a decreased expression of ICM. Decreased expression of ICM, especially PD-L1, was also observed after stimulation with GBS but not with Supplementary file1 (PPTX 1105 KB)Below is the link to the electronic supplementary material.


This work demonstrates the significance of the nanoarchitectureengineering of catalysts and sheds light on how to optimize structuresfeaturing abundant active sites and enhanced mass transfer.Mass transfer is critical in catalytic processes, especiallywhenthe reactions are facilitated by nanostructured catalysts. Strongefforts have been devoted to improving the efficacy and quantity ofactive sites, but often, mass transfer has not been well studied.Herein, we demonstrate the importance of mass transfer in the electrocatalyticoxygen reduction reaction (ORR) by tailoring the pore sizes. Usinga confined-etching strategy, we fabricate boron- and nitrogen-dopedcarbon  electrocatalysts featuring abundant active sites butdifferent porous structures. The ORR performance of these catalystsis found to correlate with diffusion of the reactant. The optimizedB,N@C with trimodal-porous structures feature enhanced O So far, electrode fabrication and device configurationdesign have been the common approach to enhance mass transfer,10 rather than tailoring the pore sizes and volumes of the nanocatalystitself.12An oftenless studied but significant factor affecting the catalyticprocess is mass transfer, which determines the efficiency of the supplyof reactants and removal of products to and from the active sitesand consequently the catalytic performance. For various gas-involvingelectrocatalysis, including hydrogen evolution reaction (HER), oxygenevolution reaction (OER), oxygen reduction reaction (ORR), electrochemicalreduction of carbon dioxide , mesopores(2–50 nm), and macropores (>50 nm) are desirable to simultaneouslyachieve large numbers of active sites and improve mass diffusion.18 Metal–organic frameworks (MOFs) are ideal precursors to obtainnanoreactors due to their customizable modular assembly and controllablemorphologic and structural evolution.22 Key pore parameters including pore shapes, sizes, and volumes canbe regulated by controlling etching conditions. By varying the etchingtime, etching temperature, and etchant concentration, mesopore sizeswere confined within a range of 2–38 nm.25 However, therange of the pore sizes is not wide enough. Furthermore, pyrolyzingzinc-based MOFs (Zn-MOFs) has been used to obtain porous metal-freecarbon materials with heteroatomic active sites.26 However, most of these active sites are deeply hidden inthe micropores of MOFs-derived nanocarbons and are unreachable forthe catalytic reaction.27 Selective etchingof MOFs followed by pyrolysis proves to be effective in obtainingtunable nanostructures where active sites are exposed to reactants.29 However, synthesizing a series of platform materials with differentporous structures but similar active sites in efficacy and quantityhas been far less developed. Therefore, the relationship between masstransfer and catalytic activity is not well understood. B,N@C nanostructureshave been derived by directly pyrolyzing B,N-containing MOF -8230 andBIF-1S31) or MOFs mixed with B-containingsubstances.40 Most of these MOF-derived B,N@C nanomaterials exhibit collapsedstructures and narrow pore size distributions.40 To obtain a great variety of pores with good controllability, thisarea needs to be studied deeply, including selecting the right precursorsand the pyrolysis conditions.Nanoreactors are effective platformmaterials with a wide rangeof structures where confined environments can modulate chemical reactions.41 For example, tannic acid cantransfer solid ZIF-8 and NH2-MIL-125(Ti) into hollow MOFsin a very short period (<30 min).41 Toaddress this issue, ammonia borane (AB), which features mild hydrolysis,was used to make the etching process controllable. Consequently, thestructure of the B,N@C nanocages was effectively tailored. These 3DB,N@Cs feature similar catalytic active sites for ORR, in terms ofchemical composition and quantity. But their performance correlateswith the porous structure, especially at the meso- and macroscale,which affects the mass transfer during the heterogeneous process.The performance of the optimized B,N@C-24 catalyst is on par withcommercial Pt/C, and the excellent catalysis is associated with thetrimodal-porosity, enabling the best combination of active site exposureand mass transfer.Herein, we developed an effectivemethod to fabricate porous three-dimensional(3D) B,N@C catalysts by sequentially etching and pyrolyzing a seriesof ZIF-8 precursors. It is known that most of the reported etchingprocesses are too fast to precisely regulate the porosity of the MOFprecursors.3BH3 + 4H2O → NH4+ + B(OH)4– + 3H2.42 Therefore, compositional and structural modifications of ZIF-8 hostswere achieved at the same time, producing AB@ZIF-xh particles, where x represents the etching time.In the subsequent pyrolysis step, AB@ZIF-xh was transformedto corresponding B,N@C-xh nanocages with hierarchicalporosities.43The synthesis of the B,N@C nanoreactors isillustrated in Figure S1). The scanning electronmicroscopy (SEM) images show negligible changes in morphologies andsizes after etching . Transmissionelectron microscopy (TEM) images reveal the structural evolution ofthe AB@ZIF-8 nanoparticles through etching . AB@ZIF-2/6/12h nanoparticles retain the solid rhombicdodecahedron structure without obvious pores as the parent ZIF-8 . Extended etching gradually led tothe formation of larger pores inside the host (The X-ray diffraction (XRD) patterns of the resultingAB@ZIF-8match the simulated powder XRD patterns based on the ZIF-8 singlecrystal S1. The s2. As illustrated by SEM images in Figure S4a, B,N@C-12h retains a polyhedral morphology similarto that of the AB@ZIF-12h precursor after pyrolysis. With the extensionin etching, B,N@C became hollow and porous with rough surfaces collapsedinward . TEM images furtherverify the correlation between the nanoarchitecture of B,N@C  in Figure S5 confirmed the decrease in micropore(<2 nm) proportions and the increase in mesopore (2–50 nm)and macropore (>50 nm) proportions during etching. N2 adsorption–desorptionisotherms were also collected to characterize the porosities of B,N@Cnanocages . 2-MIL-125(Ti) into hollow MOFs in a very short time (<30 min).41 In contrast, AB features mild hydrolysis, which makes the etchingprocess moderate and controllable. Consequently, the structure ofthe B,N@C nanocages can be effectively tailored.To study the correlation of the porosity betweenAB@ZIF-8 nanoparticlesand B,N@C nanocages, the relationship between their pore sizes andetching time was studied based on their Nncurves S6. Figurncurves S6. FigurThe NLDFT poresize distribution analysis 3e shows 48 Ramanspectra (1), 1119 (D2), 1471 (D3), and 1577(G) cm–1. D1, D2, and D3 bands correspond to the disordered carbon structure, amorphouscarbon, and carbon atoms outside of a perfectly planar graphene network,respectively, while the G band is attributed to the ordered graphitecarbon.50 The ID1/IG values for all of the B,N@C nanoreactors aresimilar, within the range of 1.44 to 1.54, indicating their similardegree of graphitization and defects. Note that the D2 andD3 bands contribute less to the ORR performance.49 Chemical compositions and chemical states wereanalyzed with XPS. Survey scans show that all of these catalysts containB, C, N, and O .High-resolution XPS spectra of B 1s can be deconvolved into threepeaks, which are assigned to B–C (190.6 eV), B–N (191.9eV), and B–O (192.5 eV) (Table S2). It is notablethat, by prolonging the etching time, the proportion of B–Oincreased significantly from 0% in B,N@C-12h to 23% in B,N@C-48h,indicating the gradual accumulation of B–O during etching.This result matches well with our proposed etching mechanism in whichthe mild hydrolysis of AB gradually releases protons, which then attack2-MIM ligands in ZIF-8. For ORR, B–O bonds are reported tohave a rather limited contribution to the performance,51 where B–C and B–N benefit theORR.53 The N 1s XPS spectra are well fitted byfive peaks corresponding to N–B (397.9 eV), pyridinic N6 (398.5eV), pyrrolic N5 (399.5 eV), graphitic N (400.8 eV), and N–O(402.4 eV). Pyridinic N6, pyrrolic N5 and graphitic N are also knownto contribute to ORR.55 The ratios of B (in B–N)to N species (in N–B) are similar in these catalysts , but the totalamounts of B and N in B,N@C-24h are slightly lower compared with theother two samples (Table S2). As shownin Table S2, all these catalysts have similar chemicalfeatures of N and all these known active sites involving B and N aresimilar in ratio. Therefore, these three catalysts with similar heteroatomicB2.5–N1 motifs can serve as an idealmaterial platform to investigate the structure-performance relationshipof nanoreactors toward catalytic reactions.B,N@C-12/24/48hwas further investigated using XRD, Raman spectroscopy,and X-ray photoelectron spectroscopy (XPS) techniques. XRD patternsin spectra 4bcan be2-saturated 0.01 M (pH 12) and 0.1 M(pH 13) KOH solution are shown in E1/2) of0.557 V vs RHE , while B,N@C-48hwith the lowest surface area shows the most negative E1/2 of 0.524 V vs RHE. These results confirm that theelectrocatalytic performance is determined by the exposure of activesites in dilute electrolytes, which is typically related to surfaceareas. In contrast, the ORR performance of the three samples in 0.1M KOH varied greatly  of 0.979 V and E1/2 of 0.861 V vs RHE, as summarized in Electrochemical ORR was used as a model reactionto test the effectof porous structural engineering on the catalytic performance andto understand the importance of mass transfer during the reaction.The performance was assessed by using a rotation ring disk electrode(RRDE) in a typical three-electrode system. The LSV disk curves collectedat 1600 rpm in an OV vs RHE S8, whileV vs RHE S8, whileV vs RHE S8, whileV vs RHE S8, whileFinite element analysis (FEA) simulations werecarried out to furtherunderstand the influence of mass transfer on the electrocatalyticperformance. According to the structural characterizations 2 and 5d,Eon, E1/2, and DLCD among these three catalysts, and its performanceis comparable to that of commercial Pt/C , which shows an Eon of 1.01V, an E1/2 of 0.867 V, and a DLCD of −5.6mA cm–2 at 0.2 mV vs RHE. Besides, the performanceof B,N@C-24 nanoreactors is on par or even better than the reportedmetal-free carbon-based catalysts (Table S3). In contrast, micropore-dominated B,N@C-12h demonstrated lower Eon, E1/2, and DLCDthan B,N@C-24h, although it has the highest heteroatom content (Table S1). This suggests that certain activesites in the micropores have no contact with the reactants, so theORR rate is limited.57 In addition, B,N@C-48hwith a larger portion of macropores also shows decreased ORR activitiescompared with those of B,N@C-24h (JK) werecalculated. B,N@C-24h with trimodal-porous distributions deliversthe highest mass-specific activity and Jk among the three catalysts , further manifestingthat the trimodal-porous nanoreactor exhibits the fastest kineticstoward the ORR. All these results prove that a trimodal-porous nanoreactoris desired for improving both mass transfer and exposure of activesites. Based on RRDE measurement  of B,N@C-24hwas calculated to be about 3.83 , indicating an efficient 4e– pathway toward ORR,which is desirable for zinc-air batteries. Similarly, electron transfernumbers were found for B,N@C-12h and B,N@C-48h.As discussed above, B,N@C-24h withtrimodal-porous structure deliveredthe largest ial Pt/C S9, whichial Pt/C S9, whichial Pt/C S9, whichWe designeda series of hierarchical porous carbon nanoreactorsdoped with B and N via efficient confined-etching and pyrolysis ofZIFs. The B,N@C nanocages have similar catalytic active sites in termsof intrinsic activity and quantity but have different pores in termsof size and volume. When tested for the ORR, the B,N@C nanoreactorwith abundant micro-, meso-, and macropores shows the highest catalyticactivity. Experimental results and FEA calculations confirm that sucha trimodal-porous architecture enhances process intensification towardORR because of enhanced mass transfer and effective active site exposure,in comparison with microporous and macroporous architectures. Thiswork demonstrates the importance of mass transfer during heterogeneouscatalysis, which should be considered when designing novel catalysts.It also proves the efficacy of the host–guest chemistry-assistedetching strategy, especially with mild etchants, for the synthesisof heteroatom-doped carbon nanoreactor systems.


Cytosine and adenosine base editors (CBEs and ABEs) are novel genome-editing tools that have been widely utilized in molecular breeding to precisely modify single-nucleotide polymorphisms (SNPs) critical for plant agronomic traits and species evolution. However, conventional BE editors are limited to achieve C-to-T and A-to-G substitutions, respectively. To enhance the applicability of base editing technology in watermelon, we developed an efficient CGBE editor (SCGBE2.0) by removing the uracil glycosylase inhibitor (UGI) unit from the commonly used hA3A-CBE and incorporating the uracil-DNA glycosylase (UNG) component. Seven specific guide RNAs (sgRNAs) targeting five watermelon genes were designed to assess the editing efficiency of SCGBE. The results obtained from stably transformed watermelon plants demonstrated that SCGBE2.0 could efficiently induce C-to-G mutations at positions C5–C9 in 43.2% transgenic plants (with a maximum base conversion efficiency of 46.1%) and C-to-A mutation at position C4 in 23.5% transgenic plants (with a maximum base conversion efficiency of 45.9%). These findings highlight the capability of our integrated SCGBE2.0 editor to achieve C-to-G/A mutations in a site-preferred manner, thus providing an efficient base editing tool for precise base modification and site-directed saturated mutagenesis in watermelon. The . .In crops, single-nucleotide polymorphisms (SNPs) are the critical genetic basis for agronomic traits , 6. ThusClALS in watermelon (Citrullus lanatus), resulting in the development of the first single-base edited herbicide-resistant watermelon variety. The watermelon CBE attained an editing efficiency of 23.0% in the T0 generation, marking a significant milestone for watermelon BE editor development [The most commonly used BE editors are CBEs and ABEs, responsible for cytosine (C-to-T) and adenine (A-to-G) base substitutions, respectively . A typicelopment .Escherichia coli tRNA-specific adenosine deaminase (ecTadA). The widely adopted ABE version is 7.10 (ecTadA-ecTadA*-nCas9) [Arabidopsis, and poplar [ABE, similar in structure to CBE, operates through a comparable mechanism. Adenosine deaminase converts adenine (A) to inosine (I), which is subsequently recognized as guanine (G) by DNA polymerase during DNA repair and replication , 13, 25.*-nCas9) , but rec*-nCas9) , 28. To d poplar –33.The optimization of BEs has primarily focused on enhancing base editing efficiency, modifying the editing window as needed, and reduction of protospacer-adjacent motif (PAM) sequence dependence , 35. ImpAlthough C-to-T and A-to-G base substitutions are well established in various plant species through CBEs and ABEs, their limited capability significantly restricts the application of base editing technology in crop molecular breeding . UNG is Arabidopsis UNG enables highly efficient C-to-G/A mutations in a site-preferred manner. This breakthrough opens up new possibilities for molecular breeding and artificial evolution not only in watermelon but also in other dicotyledonous plants.Watermelon, a highly popular summer fruit globally, is renowned for its refreshing taste and abundant nutritional value. Previous studies on the watermelon genome and genome-wide association study (GWAS) analysis have systematically elucidated the molecular mechanisms underlying trait evolution . These fArabidopsis BE studies, it was observed that CBE and ABE carrying the Ribosomal protein S5a (Rps5a) promoter, which exhibits activity in dividing cells, achieved efficient editing successfully [Arabidopsis codon-optimized hA3A-nCas9 (D10A)-UGI -UGI .ClALS1, ClDA1, and ClEOD1) were designed, and multiple transgenic watermelon plants were generated. The results revealed that both the AtUbi and 2 × 35S promoter-based CBEs achieved C-to-T editing in watermelon, with the highest base conversion efficiencies  recorded as 27.0% and 93.4%, respectively  was combined with SCGBE1.0,resulting in the development of a new editor called SCGBE2.0 (ClACC and ClARR2). A total of 81 T0 plants were obtained, and Hi-TOM deep sequencing analysis was performed. The results showed that 35 lines of these plants achieved C-to-G substitutions, representing an editing efficiency of 43.2% (35/81), and the highest efficiency is up to 66.7% (8/12) in watermelon T0 lines  of the T0 plants exhibited both C-to-T and C-to-G edits at the same position when SCGBE2.0 was used. To gain a better understanding of the increased efficiency of C-to-G editing in SCGBE2.0, a comparison of the actual C-to-T and C-to-G conversion fractions at positions C5–C9 was conducted among SCBE, SCGBE1.0, and SCGBE2.0 . The resAccording to our results, the novel hA3A-based CGBE editors successfully achieved C-to-G base substitutions. However, additional C-to-A mutations also occurred at specific positions. A total of 23.5% (19/81) of the transformants containing C-to-A mutations , primariFigure 4a andMeanwhile, a brief test was conducted to examine the PAM preference of SCGBE2.0. The results demonstrated that SCGBE2.0 successfully achieved C-to-G mutations at position C7 (15.7%) and C-to-A mutations at position C4 (15.7%) when the NAG PAM was used (sgRNA6) . HoweverBase editors, including CBE for C-to-T mutations and ABE for A-to-G mutations, have emerged as powerful gene editing tools based on CRISPR/Cas9 . These tWhile high-efficiency C-to-G editors have been established in rice, the optimization of poplar CGBE editors based on rAPOBEC1 is still needed due to their low editing efficiency in poplar T0 lines , 47. In However, the edited plants obtained using SCGBE2.0 are likely to be C to G/A/T chimeras. For chimeric mutations obtained through SCGBE2.0 editor, the first step is to conduct screening to identify the desired target mutations. This can be achieved through molecular analysis techniques such as sequencing to determine which loci possess the desired mutations. The next step is mutation evaluation, further evaluation is performed on the isolated homozygous individuals, including molecular analysis and phenotypic observations. This helps validate the stability of the mutations and assess their impact on the target traits. Additionally, it is important to consider any unforeseen mutations that may be introduced by chimeric mutations and conduct comprehensive analysis.In summary, chimeric mutations originating from SCGBE2.0 can be harnessed for the artificial evolution of watermelon genes, with the main goals being the enhancement of specific traits, improvement of gene function, augmented adaptability, or generation of novel functional variations. By engaging in iterative cycles of editing and selection, advantageous mutations can be progressively amassed, thereby facilitating gene evolution.Our findings have demonstrated that SCGBE2.0, utilizing hA3A deaminase, enables various forms of base mutations (C-to-G/A) in watermelon. However, the base conversion efficiency and range of SCGBE2.0 can still be further improved due to the limitations of deaminase and Cas protein types. The highest C-to-G conversion efficiency achieved was 46.1% at target position C7, while the highest C-to-A conversion efficiency was 45.9% at position C4. These results indicate that there is room for enhancing the base editing efficiency and expanding the editing scope of SCGBE2.0.Currently, CGBE research in dicot plants remains relatively limited, with only rAPOBEC1-CGBE being tested , 47. SimOur experiments using SCGBE2.0, which incorporates nCas9, successfully edited all targets with PAM sequences NGG and NAG. However, no editing was observed for targets with the NGA PAM sequence. The fixed PAM sequences pose a significant limitation to the application of CGBE in plants. To overcome this limitation, the utilization of Cas9 variants, such as SpCas9-VQR (NGA) and ScCas9 (NNG), which have been successfully employed in traditional base editing studies in plants, could expand the range of PAM selections for CGBE , 60. IncIn conclusion, we conducted a two-step optimization process based on hA3A-CBE to develop a CGBE editor more suitable for watermelon research and molecular breeding. Our results demonstrated that the removal of UGI and the addition of AtUNG to SCBE effectively enhanced the efficiency of C-to-G conversion. The final SCGBE2.0 editor exhibited high editing efficiencies for C-to-G mutations within the C5-C9 editing window, with the highest 46.1% efficiency observed at C7 . AdditioThe watermelon material used in this study was ‘TC’ (Tongchuan), which was collected and maintained by our research group. ‘TC’ plants were grown under natural conditions, and mature seeds were collected. Cotyledons were used as explants for watermelon transformation.Arabidopsis website (Tair: https://www.arabidopsis.org/index.jsp) to obtain the nucleotide sequences of AtUNG (sequence ID: AT3G18630), AtUbi (sequence ID: AT3G52590), and AtRps5a (sequence ID: AT3G11940). The NLS, hA3A, and nCas9 protein sequences were then optimized according to the codon preference of Arabidopsis (IDT: https://sg.idtdna.com/pages) and chemically synthesized by Sangon Biotech Co. Ltd . The fusion protein sequences were cloned into the pBSE402 vector backbone, which already contained a 35S-GFP expression cassette [To construct CBE and CGBE vectors, we performed a BLAST search on the cassette , 63.The pBSE402 vector was digested with BamHI/SacI, and the synthetic UGI and NLS fragments were amplified and inserted into the pBSE402 vector to generate UGI-NLS-E9T . After dThe pBSE402 vector was digested with BamHI/SacI, and the synthetic nCas9 and NLS fragments were amplified and inserted into the pBSE402 vector to generate nCas9-NLS-E9T. After digestion with AscI, the NLS-hAPOBEC3A fragment was amplified and inserted into nCas9-NLS-E9T to obtain the vector: NLS-hAPOBEC3A-nCas9-NLS-E9T. Finally, the promoters AtUbi and 2x35S were added to the above vector to obtain UCGBE1.0 and SCGBE1.0 vectors, respectively.The pBSE402 vector was double-digested with BamHI/SacI, and the AtUNG fragment carrying NLS was amplified and inserted into the pBSE402 vector to obtain the vector: UNG-NLS-E9T. After digestion with AscI, the nCas9 fragment was amplified and inserted into UNG-NLS-E9T to obtain the vector: nCas9-UNG-NLS-E9T. Subsequently, the NLS-hAPOBEC3A fragment was amplified and inserted into nCas9-UNG-NLS-E9T to obtain the vector: NLS-hAPOBEC3A-nCas9-UNG-NLS-E9T. Finally, the 2x35S promoter was added to the above vector to obtain the vector: SCGBE2.0.For each gene target site, sgRNA expression plasmids were constructed by amplifying the pCBC-DT1T2(Cm) vector and inserting it into the BE vectors after digestion with BsaI. SgRNA1–4 were then assembled into the CBEs, UCGBE1.0, and SCGBE1.0 vectors, respectively, while sgRNA1–7 were assembled into the SCGBE2.0 vector .All PCR fragments for plasmid construction were amplified using Phanta Max 505 Super-Fidelity DNA Polymerase . The sequences of all BE vectors and primers used in the construction can be found in Tables S1 and S2 , and all vectors are available upon request.Genetic transformation of watermelon was achieved by incubating cotyledons in diluted bacterial medium  for 14 minutes and providing with 40 mg/L acetosyringone. Subsequently, infected cotyledon explants were grown in the dark on MS solid medium containing 1.5 mg/L 6-BA for 3 days. Then, cotyledon fragments were transferred to shoot elongation medium with 200 mg/L Tilmicin and 1.5 mg/L 6-BA, which were kept in normal photoperiod conditions  for 3 weeks, and transferred to the same fresh medium every 10 days. Plants were screened for successful transformation using a hand-held lamp.http://www.hi-tom.net/hi-tom/), detailed information on mutation types and positions of each sample is provided, including read numbers, ratios, mutation types, mutation bases, and DNA sequences. Additionally, the genotype of each sample is analysed and summarized.Genomic DNA was extracted from each T0 transgenic callus line using the hexadecyltrimethylammonium bromide (CTAB) method . PCR ampWeb_Material_uhad155Click here for additional data file.


As a kind of mesenchymal-like stromal cells, endometrial regenerative cells (ERCs) have been demonstrated effective in the treatment of Concanavalin A (Con A)-induced hepatitis. However, the therapeutic mechanism of ERCs is not fully understood. Ecto-5`-nucleotidase (CD73), an enzyme that could convert immune-stimulative adenosine monophosphate (AMP) to immune-suppressive adenosine (ADO), was identified highly expressed on ERCs. The present study was conducted to investigate whether the expression of CD73 on ERCs is critical for its therapeutic effects in Con A-induced hepatitis.+ T cell co-culture assay in vitro. Besides, Con A-induced hepatitis mice were randomly assigned to the phosphate-buffered saline treated (untreated), ERC-treated, negative lentiviral control ERC (NC-ERC)-treated, and CD73-knockout-ERC (CD73-KO-ERC)-treated groups, and used to assess the CD73-mediated therapeutic efficiency of ERCs. Hepatic histopathological analysis, serum transaminase concentrations, and the proportion of CD4+ T cell subsets in the liver and spleen were performed to assess the progression degree of hepatitis.ERCs knocking out CD73 were generated with lentivirus-mediated CRISPR-Cas9 technology and identified by flow cytometry, western blot and AMPase activity assay. CD73-mediated immunomodulatory effects of ERCs were investigated by CD4+ T cells was identified in vitro. In addition, ERCs could markedly reduce levels of serum and liver transaminase and attenuate liver damage, while the deletion of CD73 on ERCs dampens these effects. Furthermore, ERC-based treatment achieved less infiltration of CD4+ T and Th1 cells in the liver and reduced the population of systemic Th1 and Th17 cells and the levels of pro-inflammatory cytokines such as IFN-γ and TNF-α, while promoting the generation of Tregs in the liver and spleen, while deletion of CD73 on ERCs significantly impaired their immunomodulatory effects locally and systemically.Expression of CD73 on ERCs could effectively metabolize AMP to ADO, thereby inhibiting the activation and function of conventional CD4Taken together, it is concluded that CD73 is critical for the therapeutic efficiency of ERCs in the treatment of Con A-induced hepatitis.The online version contains supplementary material available at 10.1186/s13287-023-03505-2. Acute ehogenies , 3, 4.Extracellular accumulation of ATP plays a pivotal role in the initiation of immunogenesis . Under p+ conventional T cells [Endometrial regenerative cells (ERCs), a kind of mesenchymal-like stromal cells derived from adult menstrual blood, exhibit the same immunomodulatory ability as mesenchymal stem/stromal cells (MSCs) and possess many advantages than MSCs , 13. We  T cells –16. Curr T cells , 18. As  T cells , 12. HowConcanavalin A (Con A)-induced hepatitis is a well-established murine model of T cell-mediated acute hepatitis . In thisERCs were isolated from adult menstrual blood collected from volunteers as described previously . BrieflyNT5E gene (code CD73) deletion in ERCs was generated with lentivirus-mediated CRISPR-Cas9 knock-out technology . SgRNA sequences targeting human NT5E were designed as follows: 1#, CACCGAACTCATCGCTCAGAAAGT; 2#, CACTTTCTGAGCGATGAGTT. The lentivirus transfection procedure was done in a biosafety cabinet with an appropriate multiplicity of infection (MOI = 30). The transfected cells were passaged at 80% confluence. After selection with 2 μg/mL puromycin , the efficiency of gene knockout was examined and subsequent experiments were performed.The The cell total protein extraction and western blotting were performed as previously described . To be s4 cells per well. The next day, the culture medium was discarded and the plates were washed three times with saline, then the Phosphate Assay Kit was used to detect the AMPase activity of these cells. In short, 200 μL of AMP -containing saline was added to each well and incubated at the temperature of 37 ℃ for 10, 30, 60, 120, and 180 min, respectively. Then, 50 μL of PiColorLock reagent mix (PiColorLock reagent and 1/100 volume of Accelerator) was added into the Pi-containing culture supernatant. Next, 5 min later, add 20 µL of the Stabilizer, then mix with the pipette. Finally, after 30 min of incubation, the plates were counted absorbance at 635 nm via the Microplate Reader .The AMPase activity of ERCs was analyzed with the help of the Phosphate Assay Kit- PiColorLock . To be specific, on the first day, ERCs, NC-ERCs, and CD73-KO-ERCs were harvested and transferred to 96-well plates for 5 × 10+ T cells were isolated from the spleen by positive sorting using the CD4 (L3T4) MicroBeads . The purity of the isolated cells was assessed by flow cytometry . Purified CD4+ T cells were cultured in RPMI-1640 medium  containing 10% FBS  and 50 IU/mL recombinant IL-2  with 5 μg/mL pre-coated anti-mouse CD3  and 3 μg/mL anti-mouse CD28 .Mouse CD44 cells/well and CD4+ T cells were seeded at 25 × 104 cells/well in 24 well plates. When indicated, AMP  was added. At the time of harvest (72 h), the cell membrane expression of the activation markers CD69 and CD154, and the dilution of CFSE as a measure of proliferation were assessed by flow cytometry. Besides that, the population of CD4+IFN-γ+ cells were detected by flow cytometry, and the IFN-γ levels in cell culture supernatants were determined by ELISA .In co-culture assay, ERCs, NC-ERCs, and CD73-KO-ERCs were seeded, respectively, at a density of 5 × 10C57BL/6 mice, 6–8 weeks old and 22–24 g weight, were used for the induction of Con A-induced hepatitis. All animals were housed in a conventional experimental environment with enough food and water following the guidelines of the China Association for the Production of Animals, and the animal experiment protocol was approved by the Animal Ethical and Welfare Committee of Tianjin Medical University General Hospital (Ethic No. IRB2022-DW-21).n = 6 per group): untreated group; ERC-treated group (naïve ERCs); NC-ERC-treated group ; and CD73-KO-ERC-treated group . As for treatments, 1 × 106 cells per mouse were administered intravenously 30 min after the injection of Con A in ERC-, NC-ERC-, and CD73-KO-ERC-treated groups. 24 h after administration of Con A, all mice were euthanized with CO2 (at a rate of 40% replacement/min in a transparent 2 L chamber for at least 10 min). Serum, liver, and spleen samples were collected and processed for further analysis.Mice were given a single injection of Con A  via the tail veins at 15 mg/kg of body weight dissolved in 200 μL of phosphate-buffered saline (PBS). For the main cell transplantation experiment, model mice were randomly assigned to the following four groups  and injected into Con A-induced hepatitis mice (these mice were independent to the main cell therapy experiments). To be specific, the cells were harvested and labeled with CM-Dil solution (2 µM) at 37 ℃ for 5 min and then at 4 ℃ for 15 min. Next, the cells were washed three times and resuspended at a density of 1 × 10For histology, livers were fixed in 10% formalin, embedded in paraffin, and sliced into 5-µm sections. Hematoxylin and eosin (H&E) staining was then conducted on sections. Confluent liver necrosis was measured on H&E-stained liver sections via ImageJ software . Immunohistochemistry was performed using antibodies for CD4 . The TUNEL assay was conducted using the One-step TUNEL FITC Apoptosis Detection Kit  according to the instructions.The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured utilizing ALT and AST assay kits  to evaluate liver function and liver injury.The flow cytometry analysis was used to identify ERCs and detect the population of T cell subsets in this study. The staining process was the same as we previously mentioned . BrieflyThe mouse IFN-γ and TNF-α ELISA kits purchased from DAKEWE  were used to detect the levels of IFN-γ and TNF-α in this study. All experimental procedures were carried out following the manufacturer's handbooks.P < 0.05; **P < 0.01; ***P < 0.001.All statistics were calculated with GraphPad Prism 9.3.0 , and the obtained experimental data were presented as mean ± SD. For the analysis of differences between multiple groups, a one-way analysis of variance (ANOVA) was used. *Knocking out CD73 on the ERCs did not change the morphology, NC-ERCs and CD73-KO-ERCs all showed a fibroblast-like phenotype as ERCs Fig. A. SuccesP < 0.001). This result further clarified the successful knockout of CD73 in CD73-KO-ERCs and the enzymatic function of CD73.To further determine the successful knockout of CD73 on ERCs, the AMPase activity of CD73 was detected indirectly utilizing the PiColorLock Phosphate Detection Reagent . As shown in Fig. + conventional T cells and added ERCs with different pretreatments. As shown in Fig. P < 0.001; CD154: P < 0.05). It is worth noticing that when exogenous AMP was introduced to the culture medium to ensure sufficient substrate for CD73 in all situations, the reduction of CD69 expression on CD4+ T cells was more apparent when cultured with NC-ERCs than CD73-KO-ERCs . There is no significant difference in the CD3+CD8+ T cell subsets between the four groups  and Th17 (CD4+IL-17A+) subsets. As shown in Fig. + T cells, as shown in Fig. +Foxp3+ Tregs, while the knockout of CD73 dampens the induction of Tregs. Taken together, the above data suggest that CD73 expression on ERCs plays a pivotal role in the regulation of local CD4+ T cell response in Con A-induced hepatitis.Excessive ATP in the extracellular component can promote the proliferation of CD4 T cells , which a+CD4+ or CD3+CD8+ T cell subsets in the spleen by flow cytometry. As shown in Fig. +CD4+or CD3+CD8+ T cells in the ERC-or NC-ERC-treated group were significantly lower than those of untreated group . Consistent with the previous results, the CD3+CD4+ T cell populations were higher in the CD73-KO-ERC-treated group when compared with NC-ERC-treated group, though CD73-KO-ERCs also perform an inhibitory effect when compared with the untreated group  and Th17 (CD4+IL-17A+) cells. As shown in Fig. P < 0.001). No obvious difference in the Th1 cell population was found between the ERC-treated group and the NC-ERC-treated group, but a significant elevation was found in the CD73-KO-ERC-treated group compared with those of the NC-ERC-treated group . Consistently, the same trend was also observed in Th17 subsets , indicating that CD73 expressing on ERCs plays a critical role in the promotion of Treg generation.Tregs were generally believed to play a significant role in the maintenance of liver self-tolerance . In the + T cell infiltration in livers, and inhibited Th1 and Th17 cell differentiation in splenocytes while promoting the generation of Tregs.Collectively, these results reveal that CD73 expression is essential for the immune regulation ability of ERCs. In Con A-induced hepatitis mice, CD73 expressing ERCs reduced CD4. untreated group, P < 0.001; TNF-α: ERC-treated group vs. untreated group, P < 0.001; serum: IFN-γ: ERC-treated group vs. untreated group, P < 0.001; TNF-α: ERC-treated group vs. untreated group, P < 0.001). The ERCs transfected with empty lentivirus did not alter the cytokine profiles both in the liver and serum compared with the ERC-treated group . However, the IFN-γ and TNF-α levels showed an obvious elevation in the CD73-KO-ERCs-treated group when compared with the NC-ERC-treated group . These results show that ERCs possess the ability to downregulate the profile of pro-inflammatory cytokines and the CD73 expression is critical for ERCs to maintain this property.To determine whether CD73 expression of ERCs plays a role in regulating cytokines in Con A-induced hepatitis, we detected the levels of IFN-γ and TNF-α in the livers and sera from different groups. As shown in Fig. + T cell activation and function in vitro and led to poor therapeutic efficiency of ERCs in Con A-induced hepatitis which was associated with extensive liver necrosis, massive infiltration of effector CD4+ T cells, decreased generation of Tregs, and elevated tissue and serum levels of pro-inflammatory cytokines .Con A-induced acute hepatitis is widely recognized as T cell-mediated hepatitis , 21, 22.NT5E gene which coded CD73 in ERCs by CRISPR/Cas-9 system, and identified using flow cytometry and western blot. In addition, the results of the CD73 enzyme function test further proved the successful knockout of CD73 on the one hand and demonstrated the high efficiency of CD73 in catalyzing AMP to ADO on the other hand.CD73 is a key enzyme in purinergic metabolism with the function of dephosphorylating immune-stimulating AMP to immune-suppressive ADO and has been demonstrated to play a key regulatory role in several pathological and physiological processes , 24. As + T cells infiltrate into the liver tissue and produce large amounts of inflammatory cytokines, such as IFN-γ, TNF-α, IL-2, and IL-6 [+ T cells both in vitro and in Con A-induced hepatitis response, while there was an apparent recovery when CD73 was knocked out on ERCs. During the occurrence of liver injury, the impaired cells release numerous amounts of ATP into the local milieus and induce the activation and immune response of CD4+ T cells [2A receptors (A2AR) expressed on T effector cells and then inhibits the excessive immune response [+ T cells is downregulated to prevent autocrine/paracrine immune-suppressive adenosine generation [+ conventional T cells in the ERC-treated and CD73-KO-ERC-treated groups.Activated T lymphocytes play a crucial role in Con A-induced liver injury –27. CD4+and IL-6 –30. In t T cells . In the  T cells , 31. As response , 32. Howneration . Conside+ T cell subsets, we have also detected the population of hepatic and splenic Tregs in different groups in the present study. Similar to our and other researchers’ previous study, ERCs could effectively enhance the population of Tregs in inflammatory conditions [. [2AR expressed on Tregs facilitated the expansion of these cells, and in turn, an increased immunoregulatory ability was acquired [Tregs are pivotal for the maintenance of immunological self-tolerance and the prevention of autoimmunity , 35. In nditions –40. Intetions [.  highlighacquired . The sigacquired . Based oThe intrahepatic IFN-γ and TNF-α are crucial proinflammatory cytokines for inducing hepatocyte apoptosis and hepatic fibrosis . Intrigu+ T conventional cells and reduced production of pro-inflammatory cytokines. On the other hand, CD73 expression of ERCs promotes the generation of immunoregulatory Tregs. However, there are also some limitations in the present study. First, due to ERCs are not suitable for monoclonal culturing, it is impossible to fully knock out CD73 in the ERCs, so there is still some CD73 expression in the CD73-KO-ERCs as shown in our flow cytometry and western blot results. These remained CD73 may partially explain the incompletely recovery in the CD73-KO-ERC-treated group when compared with NC-ERC-treated group. To solve this problem, we will consider extracting ERCs from CD73 deficient mice for further study [In the present study, some underlying mechanisms need to be discussed. On the one hand, CD73 expression improves the immunosuppressive ability of ERCs which is characterized by anti-infiltration and proliferation of pro-inflammatory CD4er study , 47. In er study , PGE-2 [er study , Gal 9 [er study , 49, andIn conclusion, CD73 is critical for ERC-mediated therapeutic efficiency in the treatment of Con A-induced hepatitis. The findings of this study serve as the foundation for the clinical use of ERCs not only in autoimmune hepatitis, but also in other immune-mediated diseases.Additional file 1: Figure S1. The purity of the isolated CD4+ cells was assessed by flow cytometry, and the percentage of live CD4+ cells achieved 97.1%.Additional file 2: Figure S2. The Full-length merge blots of CD73 and β-actin in immunoblotting (protein). The shiny images used in the manuscript corresponded to the part marked here by the black frame. The lanes on the right above were duplicates. (A) The merge blots of C73. (B) The merge blots of β-actin.Additional file 3: Figure S3. The liver infiltration of CD8+ T cells in different groups. (A) The representative pseudocolor plots were depicted. (B) show the statistical graph. One-way ANOVA was used for statistical analysis, no significance was found between those groups. Data in bar graphs represent mean ± SD.Additional file 4: Figure S4. The liver infiltration of CD4+IL-17A+ Th17 cells in different groups. (A) The representative pseudocolor plots were depicted. (B) show the statistical graph. One-way ANOVA was used for statistical analysis, no significance was found between those groups. Data in bar graphs represent mean ± SD.


This study investigates the stress corrosion cracking (SCC) behavior of type 316L stainless steel (SS316L) produced with sinter-based material extrusion additive manufacturing (AM). Sinter-based material extrusion AM produces SS316L with microstructures and mechanical properties comparable to its wrought counterpart in the annealed condition. However, despite extensive research on SCC of SS316L, little is known about the SCC of sinter-based AM SS316L. This study focuses on the influence of sintered microstructures on SCC initiation and crack-branching susceptibility. Custom-made C-rings were exposed to different stress levels in acidic chloride solutions at various temperatures. Solution-annealed (SA) and cold-drawn (CD) wrought SS316L were also tested to understand the SCC behavior of SS316L better. Results showed that sinter-based AM SS316L was more susceptible to SCC initiation than SA wrought SS316L but more resistant than CD wrought SS316L, as determined by the crack initiation time. Sinter-based AM SS316L showed a noticeably lower tendency for crack-branching than both wrought SS316L counterparts. The investigation was supported by comprehensive pre- and post-test microanalysis using light optical microscopy, scanning electron microscopy, electron backscatter diffraction, and micro-computed tomography. Additive manufacturing (AM) encompasses the technologies used to create physical objects from digital data by successively joining materials . Sinter-Stress corrosion cracking (SCC) is a form of environmentally-assisted cracking (EAC), typically nucleating from localized corrosion sites when susceptible materials are exposed above a threshold stress in specific corrosive environments ,18,19,20Austenitic stainless steel UNS S31603 (SS316L) is considered an excellent material for engineering applications due to its exceptional ductility, weldability, and corrosion resistance ,35. Its 3 to 107 K/S [Among the AM technologies, there has been a significant increase in the use of LPBF to investigate SCC in SS316L due to its ability to produce an alloy with a fully austenitic microstructure ,39,40, e 107 K/S ,49,50,51 107 K/S ,53, whic 107 K/S ,54,55. The objective of this work was to determine the SCC behavior of SS316L additively manufactured with sinter-based material extrusion when exposed to different stress levels and temperatures in an acidic chloride environment. The study focused on the impact of the sintered microstructure on the SCC initiation and crack-branching susceptibility. The SCC response was compared with those obtained from similarly tested wrought SS316L samples in solution-annealed (SA), and cold-drawn (CD) conditions. The investigation was supported by comprehensive pre- and post-test microanalyses that included light optical microscopy (LOM), scanning electron microscopy (SEM), electron backscattered diffraction (EBSD), and micro-computed tomography (micro-CT).The AM SS316L samples used in this investigation were fabricated using Bound Metal Deposition (BMD) . The technology includes the following: (i) rods of pre-alloyed SS316L powder held in a mix of polymer and wax binder, (ii) an FDM 3D printer, (iii) a solvent-based debinding unit, (iv) a sintering furnace, and (v) a cloud-based software (Live Studio v3.0) to control the process from digital object to sintered part. Further information regarding the manufacturing process can be found in a previous publication by Santamaria, R., et al. (2021) . All BMD3 cut sample using a 3D X-ray microscope with an exposure energy of 140 kV, during an exposure time of 24 h, and at a pixel resolution of 2.2 µm . The constituent phases were identified via X-ray diffraction (XRD) analysis using a Cobalt K alpha (λ = 0.179 nm) powder diffractometer radiation source operating at 35 kV 40 mA with a LynxEye detector . All XRD data were collected within 2θ ranging from 40° to 130°, using a step size of 0.015°, and a time interval of 0.7 s. Content of γ-austenite (FCC) and δ-ferrite (BCC) phases were quantified from the XRD patterns as the area of each crystalline peak over the total area of crystalline peaks. Micro-CT analysis was performed on a 2 × 2 × 2 mm2O, 10 mL HNO3, and 100 mL HCl. The concentration of nitric acid and hydrochloric acid was 70% and 32%, respectively. Microstructural characterization was conducted on representative samples, which were cut, mounted in cold epoxy resin, manually wet-ground with SiC abrasive papers, and mechanically polished down to 1 µm surface finish. Light optical microscopy (LOM) analysis was conducted on samples chemically etched with a solution containing 100 mL HElectron backscatter X-ray diffraction (EBSD) analysis was conducted on samples that were polished to a mirror surface finish with 0.02 µm colloidal silica, and then ion-milled for 30 min using a beam voltage of 8 kV at a glancing angle of 4° with full cycle rotational movements . Samples were surface-coated with a carbon film 5 µm thick to prevent electrostatic charging. Microstructures were imaged using secondary electron (SE) and backscatter (BS) detectors coupled to a field emission scanning electron microscope (FE-SEM) . Elemental composition was mapped with a high-sensitivity Oxford energy-dispersive X-ray spectroscopy (EDS) detection system attached to the FE-SEM. The content of non-metallic inclusions was quantified from EDS elemental maps by dividing the area of oxides or sulfides over the total area of the map, per ASTM E1245 . EBSD was conducted with an Oxford symmetry EBSD detector in the FE-SEM on 70° tilted samples, at a working distance of 20 mm, with a beam energy of 28 kV, and a beam current of 1 nA. A clean-up process was applied to the data to assimilate any non- or mis-indexed points, ensuring that a maximum of 10% of the points were modified. Grain boundaries were detected with a threshold misorientation of 10° in conjunction with a minimum of 8 pixels of fractional difference of misorientation variation and a kernel size of 3 by 3. Kernel average misorientation (KAM) maps were used to investigate the presence of local strain in the microstructures. This analysis was conducted using a 3 by 3 kernel size, a square kernel shape, and a maximum misorientation angle of 5°. The average grain size was measured as the maximum Feret diameter. The average grain aspect ratio was calculated as the fitted ellipse aspect ratio. Twin content was measured as the fraction length of Σ3 (<111>/60°) boundaries over the total length of γ-austenite (FCC) boundaries. The Schmid factor on the γ-austenite (FCC) phase was measured in the plane/direction {111}<110>. All data acquisition and subsequent post-processing were conducted using the software Aztec v.5.1 and AztecCrystal v.2.1.259, respectively. Tensile tests were conducted according to ASTM E8 . BMD SS3Microhardness was measured on cut samples of untested C-rings prepared similarly to the microstructural characterization procedure that removes the sample preparation induced artifacts. The samples were obtained from the middle of the uppermost curved surface of the C-ring. This test was conducted as per ASTM E384  using a 3PO4), as per ASTM G123 [The SCC susceptibility of BMD SS316L and its wrought SS316L counterparts was investigated by exposing C-ring samples to different applied stresses and temperatures. The C-rings were designed following ASTM G38 guidelines , see FigSTM G123 . In addiBMD SS316L C-rings were 3D printed in the vertical direction as shown in 2, which is twice the minimum ratio according to the ASTM G123 standard [SCC tests were conducted by immersing the C-rings in a series of Erlenmeyer flasks containing 750 mL of solution at the set constant temperatures. Each temperature was monitored regularly with a thermocouple. Duplicate specimens of stressed and unstressed BMD SS316L C-rings and their wrought counterparts were immersed in the solution. Each Erlenmeyer flask contained three different C-rings, i.e., one from each alloy stressed at the same level. The volume of solution per exposed C-ring surface area ratio was 11 mL/cmstandard . All C-rstandard . The degstandard ,63. Sizestandard .Representative BMD and wrought SS316 XRD patterns are presented in SEM-EDS analysis showed no evidence of sensitization, i.e., Cr depletion in the vicinity of the grain boundaries, in any of the SS316L alloys, as illustrated in the elemental map in Y-axis) of BMD SS316L and its wrought SS316L counterparts. The corresponding BMD SS316L {111} pole figures in Santamaria et al. (2021)  conducteThe transgranular nature of SCC in BMD SS316L was also confirmed by the EBSD analysis shown in The results from this study are consistent with the established body of knowledge showing that non-sensitized austenitic stainless steels under tensile stress are susceptible to transgranular SCC when exposed to hot acidic chloride solutions, i.e., 60 °C or above ,18,20,34The manufacturing route influenced SCC morphology. Highly branched cracks are a frequent SCC characteristic of austenitic SS in chloride solutions ,37,82. SI,SCC), an indication of their low SCC arrest capacity [The susceptibility map in capacity ,27,31,84The lower SCC resistance of BMD SS316L was attributed to the higher content of microstructural heterogeneities, such as pores and oxide inclusions, The noticeable difference in SCC morphology between wrought and BMD SS316L samples, illustrated in The weakly crystallographic textured microstructure of BMD SS316L resulted in an overall reduction of the Schmid factor, as given in SCC resistance increased in the following order: SA wrought > BMD > CD wrought SS316L.The sinter-based manufacturing process used to produce BMD SS316L resulted in lower residual stresses and lower strength, contributing to a higher SCC initiation resistance than the highly stressed CD wrought condition.The large grain aggregates, equiaxed grain morphology, weak crystallographic texture, and a large content of twin boundaries decreased the SCC crack-branching of BMD SS316L when compared to SA and CD wrought SS316L.The porosity distribution of BMD SS316L had a mixed impact on its SCC resistance. While these defects facilitated the pit-to-crack transition, they also acted as crack arrestors by blunting the crack tips.This work determined the SCC behavior of SS316L additively manufactured by sinter-based material extrusion. Tests were conducted in an acidified chloride solution  at different stress levels and temperatures to identify SCC thresholds. Results were compared with the SCC response of conventional SA and CD wrought SS316L. Results were supported by a thorough characterization that included LOM, SEM-EDS, EBSD, and micro-CT. The following conclusions were drawn based on the evidence presented above:


What are the main findings?The HCl treatment of porous carbon can generate abundant hydroxyl and carboxyl groups, while the further heat treatment can transform into thermally stable carbonyl and ether groups.Carbonyl and ether groups within porous carbon supports is beneficial to the improvement of HER performance of catalysts.What is the implication of the main finding?The surface properties of porous carbon supports can be well tuned via a HCl treatment followed by an appropriate heat treatment.The feasible improvement of HER performance by regulating surface oxygen functional groups of porous carbon supports.−2 when compared to the unmodified Pt/PC (89 mV). Pt/PC-H-700 also exhibited better durability than the Pt/PC. Overall, novel insights into the impact of the surface chemistry properties of porous carbon supports on the HER performance of Pt/C catalysts were provided, which were useful for highlighting the feasible improvement of HER performances by regulating the surface oxygen species of porous carbon supports.The oxygen functionalization of carbon materials has widely been employed to improve the catalytic performance of carbon-supported Pt (Pt/C) catalysts. Hydrochloric acid (HCl) has often been employed to clean carbons during the preparation of carbon materials. However, the effect of oxygen functionalization through a HCl treatment of porous carbon (PC) supports on the performance of the alkaline hydrogen evolution reaction (HER) has rarely been investigated. Herein, the impact of HCl combined with the heat treatment of PC supports on the HER performance of Pt/C catalysts has been comprehensively investigated. The structural characterizations revealed similar structures of pristine and modified PC. Nevertheless, the HCl treatment resulted in abundant hydroxyl and carboxyl groups and the further heat treatment formed thermally stable carbonyl and ether groups. Among the catalysts, Pt loading on the HCl-treated PC followed by a heat treatment at 700 °C (Pt/PC-H-700) exhibited elevated HER activity with a lower overpotential of 50 mV at 10 mA cm Mosemission . This isoduction ,6,7.H*) [1/NMHCS) is much more active and stable than the commercial 20% Pt/C and Pt nanoparticle (PtNP) counterparts for the HER [−2, a high mass activity of 2.07 A mg−1Pt at a 50 mV overpotential, a low Tafel slope of 56 mV dec−1, and an excellent durability in acidic electrolyte. Owing to the high dispersion of Pt, its hollow structure, large surface area, and nitrogen dopant and hierarchical pore features, a nitrogen-doped hollow porous carbon polyhedrons-supported dispersed Pt nanoparticles (Pt/NHPCP) catalyst exhibited an enhanced HER activity and durability when compared to commercial Pt/C [−2 and a Tafel slope of 27 mV dec−1, much lower than that of commercial Pt/C (69.9 mV and 32 mV dec−1). Similarly, a functionalized multiwall carbon-nanotubes-supported Pt single-atom and cluster (Pt/f-MWCNTs) catalyst exhibited outstanding mass activity and stability for catalyzing the HER in an acid medium, due to the unique surface properties of its carbon surface [1-O2-Fe1-N4), the Pt1@Fe-N-C electrocatalyst exhibited a high catalytic activity toward the HER with a low overpotential of 60 mV at 10 mA cm−2 and a small Tafel slope of 42 mV dec−1 in acidic media [Platinum (Pt) is the most efficient electrocatalyst for the HER, owing to its favorable Gibbs adsorption-free energy of H* (∆GH*) ,8,9. HowH*) ,11,12,13H*) ,15. ThesH*) , which hH*) ,17,18. GH*) . In this the HER . This caial Pt/C . The Pt/ic media .3, H2SO4, KOH, H3PO4, HF, C6H6, and H2O2 treatments [3 and H2O2 may introduce abundant surface oxygen functional groups on carbon supports to disperse/anchor their Pt nanoparticles and enhance their metal–support interactions, thereby improving the catalytic performances of the catalysts. However, different oxidation reagents often result in different electrochemical performance electrocatalysts. For example, Pt/C catalysts synthesized by the H2O2 treatment of carbon supports have exhibited a higher cycling stability than those prepared by a HNO3 treatment [6H6- or H3PO4-treated CB-supported Pt catalysts [3-treated carbon-black-supported Pt catalysts have depicted higher electrochemically active surface areas, better oxygen reduction reaction activities, and lower stabilities than unmodified carbon-black-supported Pt catalysts [In general, the structural and surface properties of carbon supports can significantly influence the states of supported Pt species, thereby regulating the catalytic performance of Pt/C catalysts. Chemical treatment can regulate the surface chemistry properties of these carbon supports, especially their oxygen functionalization, which can improve the catalytic performance of the Pt/C catalysts. For instance, the surface properties of carbons can be changed by HNOeatments ,23,24,25reatment . KOH-treatalysts . Therefoatalysts ,28,29,30−2 when compared to the unmodified Pt/PC (89 mV). Meanwhile, the catalytic stability of the Pt/PC-H-700 was better than that of the Pt/PC from the durability measurements.Herein, porous carbon (PC) derived from coconut shell was utilized as a support for preparing a series of Pt/C catalysts. The effects of the HCl treatment coupled with a further heat treatment on the HER performances of Pt/C catalysts were elaborately investigated. The structural characterizations revealed the similar porous structures of the pristine PC and modified PC. However, the HCl treatment induced abundant oxygen functional groups, such as hydroxyl and carboxyl groups, while the subsequent heat treatment formed thermally stable oxygen groups such as carbonyl and ether groups. Among the prepared samples, the optimized Pt/PC-H-700 displayed an improved HER catalytic activity, with a lower overpotential of 50 mV at 10 mA cm−1. In this reaction, the mass ratio of steam to carbon was fixed at 3:1. Afterward, the PC (10 g) and hydrochloric acid  were added into a round-bottomed glass flask, followed by mounting in an oil bath device and boiling (100 °C) for 5 h. The obtained product after the HCl treatment was taken out, cooled, washed with deionized water until it reached a neutral pH, and dried in a vacuum oven at 60 °C for 12 h to yield the PC-H samples.The PC used in this work was derived from coconut shell and prepared by the water–gas activation method. Typically, the coconut shell was first directly carbonized at 450 °C for 1 h and then activated by water steam at 880 °C for 2.5 h, at a heating rate of 10 °C min−1. Briefly, the as-obtained PC-H (1 g) was placed into a tube furnace and heated at a rate of 5 °C min−1 from room temperature to the target temperature  for 2 h, before being cooled down to room temperature to yield PC-H-500, PC-H-600, PC-H-700, and PC-H-800, respectively.The annealing treatment of the PC-H was performed under an Ar atmosphere at a flow rate of 50 mL min2PtCl6·6H2O, 39 µL) solution at a Pt concentration of 63.885 mg mL−1 was then slowly added dropwise and stirred for 30 min before heating at 140 °C for 3 h in an oil bath. After being cooled down to room temperature, the prepared catalyst was taken out and washed with ethanol and deionized water until it reached a neutral pH, followed by drying at 60 °C for 12 h to yield the Pt/PC catalyst. The other catalysts, such as Pt/PC-H, Pt/PC-H-500, Pt/PC-H-600, Pt/PC-H-700, and Pt/PC-H-800, were obtained through the same preparation process using PC-H, PC-H-500, PC-H-600, PC-H-700, and PC-H-800 as supports, respectively.The Pt/C catalyst was obtained by a modified polyol reduction method, with the Pt loading content fixed at 5 wt.% ,32. TypiThe surface morphologies and elemental distributions of the carbon supports were studied by scanning electron microscopy  equipped with energy dispersive spectroscopy (EDS). The sizes and distributions of the Pt nanoparticles were visualized by transmission electron microscopy . The crystalline structures of the samples were identified by X-ray diffraction  equipped with monochromated Cu Kα1 radiation (λ = 1.5418 Å). The defect information of each porous carbon was obtained by Raman spectroscopy (Thermo Fisher DXRxi) equipped with a laser source of 532 nm. The surface oxygen functional groups of the carbons were examined by Fourier transform infrared spectroscopy . The surface chemical compositions of the samples were identified by X-ray photoelectron spectroscopy  equipped with an Al anode (Al Kα = 1486.68 eV). The Ar adsorption–desorption isotherms of the carbon supports were obtained on a Quantachrome surface area analyzer (Autsorb IQ-2) at 87 K. The specific surface areas were calculated by the Brunauer–Emmett–Teller (BET) equation and the pore size distributions were determined by the quenched solid density functional theory (QSDFT), based on a slit-pore model.−2. The working electrodes were obtained by carefully dropping 10 µL of homologous catalyst ink, which was prepared by mixing 3 mg of the catalyst and 50 µL of a 5 wt% Nafion solution in 450 µL of anhydrous ethanol, sonicated for 0.5 h on a GC surface and followed by drying in air.The electrocatalytic performances were tested on an electrochemical workstation (Ivium) connected to a typical three-electrode cell filled with 1 M KOH electrolyte at room temperature. An Ag/AgCl electrode (3.5 M KCl) and graphite electrode were employed as the reference and counter electrodes, respectively. The working electrodes consisted of an L-shaped glassy carbon (GC) substrate with a diameter of 5 mm and a geometric area of 0.196 cm−1. The reaction kinetics were examined by electrochemical impedance spectroscopy (EIS) at an overpotential of 90 mV and amplitude of 5 mV (105 Hz–0.1 Hz). The accelerated durability test (ADT) was evaluated using 5000 cyclic voltammetry (CV) profiles from −0.8 V to −1.3 V at 100 mV s−1. Long-term chronopotentiometry (V-t) measurements were conducted at 10 mA cm−2 for 10 h. Time-dependent current density (I-t) profiles were collected at a fixed overpotential of 110 mV.The HER catalytic activity was studied by linear sweep voltammetry (LSV) at a scan rate of 2 mV sThe synthesis process of the Pt/PC-H-T catalyst is schematically illustrated in The structural characteristics of the as-prepared supports were investigated by various analytical methods. The SEM morphologies of the PC, PC-H, and PC-H-700 supports are depicted in 0 < 0.01) suggested the presence of abundant micropores in all the supports. The continuous rise in the high P/P0 range and the existence of H4-type hysteresis loops (0.4 < P/P0 < 0.98) demonstrated the presence of mesopores due to the capillary condensation phenomenon of the liquid Ar in mesopores. These results confirmed the formation of a hierarchical porous structure in all the carbon supports, which was conducive to providing abundant active sites for Pt dispersion and anchoring, as well as offering adequate catalyst/electrolyte interfaces for charge/ion accumulation. The QSDFT pore size distributions of all the carbon supports are displayed in The porosity of each support was examined with the Ar adsorption–desorption isotherms and the results of PC, PC-H, PC-H-500, PC-H-600, PC-H-700, and PC-H-800 are provided in −1 and ~1589 cm−1, which were ascribed to the D-band  and G-band , respectively [−1, corresponding to the I-, D-, D″-, and G-bands, respectively. In general, the intensity ratio between the D-band and G-band (ID/IG) can be utilized to quantify the defect densities or graphitization degrees of carbon materials [D/IG values of the PC, PC-H, PC-H-500, PC-H-600, PC-H-700, and PC-H-800 supports were determined to be 2.38, 2.32, 2.38, 2.31, 2.34, and 2.39, respectively. Therefore, the HCl treatment followed by the heat treatment did not drastically change the defect density of the PC  in the hydroxyl groups, carboxyl groups, or adsorbed water [−1 belonged to the C=C skeletal vibration of the aromatic rings (ν C=C) [−1 was carefully investigated. For the unmodified PC, the characteristic band of the oxygen functional groups looked very faint. The peak centered at 1385 cm−1 can be attributed to the bending vibrations of the O-H (δ O-H) [−1 to 900 cm−1 may be ascribed to the C-O vibration. After the HCl treatment, the peak intensity of the PC-H at ~1089 cm−1 (ν C-O) obviously increased, suggesting enhanced oxygen functional groups on the PC surface. The further heat treatment of the PC-H resulted in the emergence of two peaks located at ~1168 cm−1 and ~1078 cm−1, which were attributed to the stretching vibration of C-O-C (ether) and C-O (ν C-O), respectively [−1 (ν C-O-C) appeared in the PC, PC-H-500, PC-H-600, PC-H-700, and PC-H-800 [−1 also emerged at heating temperatures of over 600 °C, which was ascribed to the stretching vibration of C=O (ν C=O) [The surface oxygen functional groups of the carbon supports were examined by an FTIR analysis. As described in ed water ,42. The  (ν C=C) ,44. To i (δ O-H) ,45. The ectively ,46. A pePC-H-800 . Another (ν C=O) ,48. Ther (ν C=O) ,44.2 C=C content of the PC decreased after the HCl treatment, leading to rising trends of the C-O, C=O, and COOH contents. The subsequent heat treatment of the PC resulted in a gradual enhancement of the sp2 C=C content, while declining the C-O, C=O, and COOH contents. A further analysis of the high-resolution O 1s deconvolution spectra revealed more intuitive components of the oxygen functional groups. In The surface chemistry of the carbon supports was investigated by XPS. As illustrated in 0 4f7/2 and Pt0 4f5/2), (Pt2+ 4f7/2 and Pt2+ 4f5/2), and (Pt4+ 4f7/2 and Pt4+ 4f5/2), respectively [δ+ (such as Pt2+ and Pt4+) species, accounting for 65–69% of the total number of the Pt nanoparticles. This could be attributed to the electron transfer process from the Pt to the carbon supports, thanks to the higher electronegativity of the C and O when compared to the Pt. On the other hand, the oxophilic microscopic Pt nanoparticles formed a dissolution-inhibiting passivation layer of PtOx, which was useful for increasing the number of Ptδ+ species and improving the catalyst’s stability [After loading the Pt on the carbon supports, the existential states of the Pt in the Pt/PC, Pt/PC-H, and Pt/PC-H-700 catalysts were studied by XRD. As shown in ectively ,12,16. Ttability . The SEMThe distributions and sizes of the Pt nanoparticles were visualized by TEM/HRTEM and the results of the Pt/PC and Pt/PC-H-700 catalysts are gathered in The Pt particle size distribution was acquired by measuring the average sizes of the 150 Pt nanoparticles from the TEM images of the Pt/PC and Pt/PC-H-700 catalysts . The Pt/−2, values lower than those of the Pt/PC (89 mV), Pt/PC-H (79 mV), Pt/PC-H-500 (75 mV), Pt/PC-H-600 (65 mV), and Pt/PC-H-800 (73 mV) catalysts. Furthermore, the Pt/PC-H-700 catalyst also exhibited a smaller Tafel slope of 29.4 mV dec−1 than the Pt/PC (49.8 mV dec−1), Pt/PC-H (46.3 mV dec−1), Pt/PC-H-500 (44.8 mV dec−1), Pt/PC-H-600 (38.7 mV dec−1), and Pt/PC-H-800 (40.0 mV dec−1) catalysts  in the Nyquist plot represents the equivalent series resistance (Rs), consisting of the electrolyte resistance, catalyst resistance, contact resistance of the electrode–catalyst interface, and interface resistance of the catalyst–electrolyte interface [The HER reaction kinetics under polarization conditions and a constant overpotential of 90 mV were explored by EIS and Nyquist plots of the Pt/PC, Pt/PC-H, Pt/PC-H-500, Pt/PC-H-600, and Pt/PC-H-800 catalysts, which are given in nterface ,62. On tnterface ,49. Amon2 bubbles generated during the initial HER process. Among the catalysts, Pt/PC-H-700 showed the highest initial current density of 47.0 mA cm−2 when compared to Pt/PC (26.4 mA cm−2) and Pt/PC-H (29.2 mA cm−2). After 10 h of the continuous HER process, Pt/PC-H-700 still exhibited a higher current density of 11.6 mA cm−2 than Pt/PC (3.6 mA cm−2) and Pt/PC-H (4.5 mA cm−2). Meanwhile, Pt/PC-H-700 displayed slightly slower attenuation in its current density with a 24.69% retained current density, superior to that of Pt/PC and Pt/Pt/PC-H, with current density delays of 13.66% and 15.34%, respectively. Thus, Pt/PC-H-700 possessed a better HER durability than Pt/PC and Pt/PC-H. The HER stability features of the Pt/PC, Pt/PC-H, and Pt/PC-H-700 catalysts were studied by ADT for 5000 continuous CV cycles. The LSV curves of the Pt/PC, Pt/PC-H, and Pt/PC-H-700 catalysts before and after the 5000 CV sweeps are presented in 10 decay of 10 mV than the Pt/PC (28 mV) and Pt/PC-H (10 mV), demonstrating a superior cycling stability. As depicted in −2 when compared to Pt/PC and Pt/PC-H. Therefore, the I-t, ADT, and V-t results all pointed to the excellent HER stability of Pt/PC-H-700 when compared to Pt/PC and Pt/PC-H, owing to the formation of thermally stable oxygen functional groups.The HER durability features of the Pt/PC, Pt/PC-H, and Pt/PC-H-700 catalysts were evaluated through I-t scanning at a fixed overpotential of 110 mV. As illustrated in Overall, the structural analyses and HER electrocatalytic performances revealed the benefit of thermally stable oxygen functional groups, such as ether groups and carbonyl groups, on carbon supports in the dispersion and anchoring of Pt nanoparticles, as well as the enhancement of the interfacial interaction between the carbon supports and catalysts, leading to improved HER catalytic performances of the catalysts. −2, better than those of the Pt/PC (89 mV) and Pt/PC-H (79 mV) catalysts. According to V-t, I-t, and ADT analyses, the Pt/PC-H-700 catalyst also displayed a better HER stability than the Pt/PC and Pt/PC-H catalysts. In sum, the interaction between the Pt nanoparticles and porous carbon supports could be improved by optimizing the surface chemistry properties of the carbon supports. In other words, the surface properties of porous carbon supports may be modified by a HCl treatment followed by an appropriate heat treatment, which is suitable for the rational optimization and regulation of the oxygen functional group species on porous carbon supports for enhanced HER performances.The role of oxygen functionalization via a HCl treatment of porous carbon supports on the HER performance of Pt/C catalysts was successfully clarified. After the HCl treatment followed by a heat treatment, the structural characterizations showed negligible changes in the morphologies, specific surface areas, pore volumes, and microcrystallines of the supports. However, the HCl treatment introduced enriched hydroxyl and carboxyl groups, while the further heat treatment transformed the functional groups into thermally stable carbonyl and ether groups. As a result, the optimized Pt/PC-H-700 catalyst exhibited an elevated HER activity with a low overpotential of 50 mV at 10 mA cm