De novo design of a nanoregulator for the dynamic restoration of ovarian tissue in cryopreservation and transplantation.

The cryopreservation and transplantation of ovarian tissue underscore its paramount importance in safeguarding reproductive capacity and ameliorating reproductive disorders. However, challenges persist in ovarian tissue cryopreservation and transplantation (OTC-T), including the risk of tissue damage and dysfunction. Consequently, there has been a compelling exploration into the realm of nanoregulators to refine and enhance these procedures. This review embarks on a meticulous examination of the intricate anatomical structure of the ovary and its microenvironment, thereby establishing a robust groundwork for the development of nanomodulators. It systematically categorizes nanoregulators and delves deeply into their functions and mechanisms, meticulously tailored for optimizing ovarian tissue cryopreservation and transplantation. Furthermore, the review imparts valuable insights into the practical applications and obstacles encountered in clinical settings associated with OTC-T. Moreover, the review advocates for the utilization of microbially derived nanomodulators as a potent therapeutic intervention in ovarian tissue cryopreservation. The progression of these approaches holds the promise of seamlessly integrating nanoregulators into OTC-T practices, thereby heralding a new era of expansive applications and auspicious prospects in this pivotal domain.

An integrated machine learning framework for a discriminative analysis of schizophrenia using multi-biological data.

Finding effective and objective biomarkers to inform the diagnosis of schizophrenia is of great importance yet remains challenging. Relatively little work has been conducted on multi-biological data for the diagnosis of schizophrenia. In this cross-sectional study, we extracted multiple features from three types of biological data, including gut microbiota data, blood data, and electroencephalogram data. Then, an integrated framework of machine learning consisting of five classifiers, three feature selection algorithms, and four cross validation methods was used to discriminate patients with schizophrenia from healthy controls. Our results show that the support vector machine classifier without feature selection using the input features of multi-biological data achieved the best performance, with an accuracy of 91.7% and an AUC of 96.5% (p < 0.05). These results indicate that multi-biological data showed better discriminative capacity for patients with schizophrenia than single biological data. The top 5% discriminative features selected from the optimal model include the gut microbiota features (Lactobacillus, Haemophilus, and Prevotella), the blood features (superoxide dismutase level, monocyte-lymphocyte ratio, and neutrophil count), and the electroencephalogram features (nodal local efficiency, nodal efficiency, and nodal shortest path length in the temporal and frontal-parietal brain areas). The proposed integrated framework may be helpful for understanding the pathophysiology of schizophrenia and developing biomarkers for schizophrenia using multi-biological data.

Divergent Alterations of Structural-Functional Connectivity Couplings in First-episode and Chronic Schizophrenia Patients.

Emerging evidence suggests that the coupling relating the structural connectivity (SC) of the brain to its functional connectivity (FC) exhibits remarkable changes during development, normal aging, and diseases. Although altered structural-functional connectivity couplings (SC-FC couplings) have been previously reported in schizophrenia patients, the alterations in SC-FC couplings of different illness stages of schizophrenia (SZ) remain largely unknown. In this study, we collected structural and resting-state functional MRI data from 73 normal controls (NCs), 61 first-episode (FeSZ) and 78 chronic (CSZ) schizophrenia patients. Positive and negative syndrome scale (PANSS) scores were assessed for all patients. Structural and functional brain networks were constructed using gray matter volume (GMV) and resting-state magnetic resonance imaging (rs-fMRI) time series measurements. At the connectivity level, the CSZ patients showed significantly increased SC-FC coupling strength compared with the FeSZ patients. At the node strength level, significant decreased SC-FC coupling strength was observed in the FeSZ patients compared to that of the NCs, and the coupling strength was positively correlated with negative PANSS scores. These results demonstrated divergent alterations of SC-FC couplings in FeSZ and CSZ patients. Our findings provide new insight into the neuropathological mechanisms underlying the developmental course of SZ.

Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy.

DDX43 promoter is frequently hypomethylated and may predict a favorable outcome in acute myeloid leukemia.

DEAD box polypeptide 43 (DDX43), a cancer/testis antigen (CTA), has been found to be overexpressed in various solid tumors and some hematologic malignancies. In the present work hypomethylation of the DDX43 gene was detected in 15% (32/214) of primary acute myeloid leukemia (AML) using real-time quantitative methylation-specific PCR (RQ-MSP). The level of DDX43 expression was correlated with DDX43 hypomethylation (R=0.277, P=0.014). Moreover, bisulfite sequencing confirmed the significant correlation between the methylation density and the level of DDX43 hypomethylation. Additionally, restoration of DDX43 expression in the K562 cell line by 5-aza-2'-deoxycytidine treatment confirmed a direct contribution of methylation in regulating the DDX43 gene. DDX43 hypomethylation was observed more frequently in favorable group (21.4%) and intermediate group (15.8%) than in poor group (0%) (P=0.009). AML patients with DDX43 hypomethylation had a better overall survival (median not obtained) than those with DDX43 methylation (median 8 months, 95% confidence interval 5.6-10.4 months) (P=0.014). In summary, the DDX43 gene is activated by promoter hypomethylation and DDX43 hypomethylation may be a favorable prognostic factor in AML.

Gene therapy in B-NHL cell line using adenovirus-mediated transfer of secretable trimeric TRAIL gene expression driven by CD20 promoter.

CD20 is a crucial target to B-non-Hodgkin lymphoma (NHL), in fact, a humanized anti-CD20 monoclonal antibody, rituximab, is widely applied in clinical practice. However, resistance to rituximab often occurs in B-NHL patients, which has encouraged us to find new medications to treat B-NHL. In this study, we designed a gene therapy strategy targeting CD20 at a transcriptional level mediated by adenovirus, in which the stTRAIL gene was driven by a specific CD20 promoter fragment. We cloned the CD20 promoter from genome DNA of BJAB cell, a CD20-positive cell line, and identified its specific transcriptional activity with a dual-luciferase reporter assay system. Meanwhile, we constructed the stTRAIL gene sequence, which contained secretion signal, isoleucine zipper, and soluble TRAIL gene sequence, in which the isoleucine zipper facilitated the product of this gene sequence to form a functional homotrimer. The recombinant adenovirus was termed as AdP20-stTRAIL, which carried on the fused gene of the CD20 promoter fragment and the stTRAIL gene. Our studies confirmed that the stTRAIL could be expressed and secreted from BJAB cells infected with AdP20-stTRAIL specifically, and it inhibited the growth of these infected BJAB cells in vitro and in vivo. Our results indicate that the gene therapy using stTRAIL gene driven by a CD20 promoter may be an effective strategy in B-NHL treatment.

[Preparation and identification of monoclonal antibody against human Flt-1].

AIM: To prepare functional monoclonal antibodies(mAb)against recombinant human Flt-1(rhFlt-1). METHODS: A cell line stable secreting mAb was established by using FLT-1 extracellular domain III as antigen and hybridoma technique. Then it was purified in large-scale from mouse ascites by protein G affinity chromatography. The characteristics of mAb were then determined by ELISA, Western blotting and FACS. RESULTS: The immunoglobin subtype of mAb XA12 was IgG1 with kappa (κ) light chains, and it could recognize rhFlt-1 specifically. Furthermore, mAb XA12 could bind to rhFlt-1with high affinity (K=1.28±0.05 nmol/L). It could also be used to detect Flt-1-positive cells, such as human umbilical vascular endothelial cells (HUVECs) and K562/A02 in a dose-dependent fashion. CONCLUSION: A hybridoma cell line secreting functional anti-rhFlt-1 mAb was successfully prepared. The antibody can be used to study the function of Flt-1 and further potentially optimized for clinical purpose.

[In vivo imaging analysis of biodistribution of FITC-labeled Rituximab in lymphoma-bearing nude mice].

OBJECTIVE: To conduct an in vivo optical imaging analysis of the biodistribution of antibody Rituximab in lymphoma tumor-bearing nude mice. METHODS: Laser scanning confocal microscope and flow cytometry were employed to determine the affinity of FITC-labeled Rituximab (FITC-Rituximab) with human lymphoma Raji cells. And the in vivo optical imaging system was used to analyze the biodistribution of FITC-Rituximab in lymphoma-transplanted xenograft nude mice. RESULTS: The results of flow cytometry and laser scanning confocal microscope demonstrated that FITC-Rituximab had remarkable affinity with lymphoma Raji cells and was mainly bound at cell membrane. The results of in vivo imaging analysis suggested that FITC-Rituximab could specifically accumulated at peritumor tissue less than 1 h, then penetrated into the interior of tumor and concentrated in 3-4 h. And the specific concentration of FITC-Rituximab could still been observed more than 8-10 h whereas there was no apparent fluorescence at other tissues. Furthermore, the results observed from a two-flank tumor xenograft model showed that FITC-Rituximab possessed specific binding affinity for CD20-overexpressed lymphoma. CONCLUSION: The in vivo optical imaging system can accurately monitor the distribution of FITC-Rituximab in tumor-bearing nude mice. And this technique has a reference value and significance for a real-time analysis of tumor-targeting capability of antibody drugs.

[Effect of calmodulin antagonist O-4-ethoxyl- butyl-berbamine on the proliferation of human breast cancer cell line MCF-7 and its relevant mechanism].

OBJECTIVE: To investigate the effect of calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine (EBB) on proliferation of human breast cancer cell MCF-7 and its possible mechanism. METHODS: MTT assay was used to analyze the effect of EBB on tumor cells growth. Flow cytometry was used to detect its impact on the cell cycle of MCF-7 cells. Immunofluoresce labeling technique and laser scanning confocal microscope were used to reveal the changes of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum in the cells. RESULTS: The IC50 value of EBB in MCF-7 cells was (13.0 +/- 3.7) micromol/L. MCF-7 cells were arrested at S phase after EBB treatment. Meanwhile, depolymerization of the microtubule and microfilament, impairment of the mitochondrion and swelling of endoplasmic reticulum were observed. CONCLUSION: EBB arrests MCF-7 cells at S phase by inhibiting the growth of MCF-7 cells, which may be related to the changes of structures and functions of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum.

[The influence of Ara-C on anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.].

OBJECTIVE: To investigate the role of Ara-C in regulating anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells. METHODS: The diabody of anti-CD3/anti-Pgp was purified by E-tag affinity chromatography. K562 and K562/A02 cells were treated with Ara-C. The expressions of B7-1 and B7-2 on K562 and K562/AO2 cells were detected by FACS. The cytotoxicity of T-lymphocytes combined with anti-CD3/anU-Pgp plus Ara-C was analyzed by CytoTox 96 nonradioactive method. RESULTS: The expressions of B7-1 and B7-2 on K562 and K562/A02 cells treated by Ara-C was significantly higher than those untreated. The effect/target ratio was from 0.39:1 to 25:1, and the killing rate of activated T cells to anti-drug-resistant leukemia cells was from (16.44 +/- 1.20)% to (60.49 +/- 2.90)%. The killing rates were increased gradually, with both the effect/target ratio and the antibody concentration increasing (P < 0.05). CONCLUSION: Ara-C may be an important adjuvant for improving anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.

[Three-step purification of preparative-scale antiCD20 (Fab')2].

OBJECTIVE: To establish a three-step purification method of preparative-scale antiCD20 (Fab')2 using AKTA prime. METHODS: AntiCD20 (Fab')2 was extracted by hyperosmotic solution and then purified by CM sepharose FF, phenyl sepharose FF, and protein G sepharose FF. RESULTS: Around 8 mg anti-CD20 (Fab')2, whose purification was 96.678%, was purified. The antigen-binding activity of antiCD20 (Fab')2 was similar to that of antiCD20 (Fab')2 purified by protein G sepharose FF and S-100. CONCLUSION: The three-step purification method can obtain high-purity preparative-scale antiCD20 (Fab')2 in a simple way.

[Preparation and activity detection of monoclonal antibody against anti-CD3 ScFv].

OBJECTIVE: To prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration. METHODS: McAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb. RESULTS: McAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively. CONCLUSION: The McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.

[Construction and expression of human 4-1BBL/anti-CD20 fusion protein].

AIM: To construct and express human 4-1BBL/anti-CD20 bispecific fusion protein and identify its biological activity. METHODS: PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispecific fusion protein. DNA sequencing was performed by the terminus of the fusion protein nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS. RESULTS: The data of DNA sequence showed that human 4-1BBL/anti-CD20 bispecific fusion protein was correct. The fusion protein was recovered in high yield (up to 4 mg/L) after E-tag purification and predominantly(90%) as a dimer. The fusion protein could bind to Raji cells(CD20(+)) and A549 cells(4-1BB(+)), respectively. CONCLUSION: The human 4-1BBL/anti-CD20 bispecific fusion protein with high level expression was successfully obtained and could bind to Raji ceIls and A549 cells.

[Up-regulation of CD86 and cytokine expression in acute leukemia cells by Ara-C].

AIM: To observe the effects of Ara-c on the expression of CD86 molecule on acute leukemia cells and explore the possible mechanisms. METHODS: The expression of CD86 on U937, HL-60 and NB4 cell lines treated with or without Ara-C wa assayed by flow cytometry. The mRNA expression of CD86, NF-kappaB, IFN-gamma was examined by semiquantitative RT-PCR. RESULTS: UP-regulation of CD86 was observed on those cells treated by Ara-c. The level of CD86 and NF-kappaB mRNA in Ara-c treated cells was significantly enhanced. IFN-gamma was detectable 72 hours after T cell activation. CONCLUSION: Ara-C can enhance CD86 and NF-kappaB expression on acute leukemia cells, which may play critical role in T cell activation and differentiation.

[The role of extracellular domain of human 4-1BBL in regulating activities of human peripheral blood lymphocytes in vitro].

AIM: To investigate the role of the extracellular domain of human 4-1BBL (ex4-1BBL) in regulating the in vitro activities of peripheral blood lymphocytes (PBL). METHODS: Viable cells were quantified with Trypan-blue exclusion assay. CytoTox 96 Nonradioactive Cytotoxicity Assay Kit was used for measuring LDH levels of supernatant. ELISA kit was used for measuring IL-2 level. In vitro cytotoxity of PBL combined with anti-CD3/anti-Pgp bispecific diabody plus ex4-1BBL was analyzed with CytoTox 96 nonradioactive method. RESULTS: Ex4-1BBL can increase the proliferation of PBL, reduce cell death, promote IL-2 secretion, and the experimental group with ex4-1BBL showed obviously enhanced cytotoxic effect toward K562/A02 cells. CONCLUSION: Ex4-1BBL may be an important adjuvant for improving activities of PBL.

[Mechanism of synergistic anti-tumor effect of PH II -7 with doxorubicin on multi-drug resistant HL-60/ADR cells].

OBJECTIVE: To study the synergistic mechanism between PH II -7 and doxorubicin against multi-drug resistant HL-60/ADR cells and its parent HL-60 cells. METHODS: The anti-tumor activity of doxorubicin alone and combined with PH II -7 were measured by MTT assay. RNA was extracted from the cells treated with PH II -7 for different times or doses then the expression of MRP gene was measured by RT-PCR. Confocal laser scanning microscopy and FACS were used to detect the intracellular cumulation of doxorubicin in PH II -7 treated HL-60 and HL-60/ADR cells. RESULTS: PH II -7 has anti-tumor effect with IC50 of (0.83 +/- 0.08) micromol/L and (1.74 +/- 0.56) micromol/L for HL-60 and HL-60/ADR, respectively. It could potentiate the anti-tumor effect of doxorubicin with CDI of 0.7 and 0.43 for HL-60 and HL-60/ADR, respectively. PH II -7 and doxorubicin act synergistically in inhibiting the proliferation of HL-60 and HL-60/ADR cells and down-regulating the expression of MRP gene in a dose and time dependent manner. PH II -7 restored the intracellular cumulation of doxorubicin in HL-60/ADR cells to 55% of that in HL-60 cells. CONCLUSION: PH II -7 can significantly hasten the cytotoxicity of doxorubicin to HL-60 and HL-60/ADR cells through down-regulating the expression of MRP. The synergistic effect was more obvious in HL-60/ADR cells.

[Construction and expression of diabody [CD3 x Pgp] without Etag].

AIM: To construct and express a diabody [CD3 x Pgp] without Etag and analyse its biological activity. METHODS: In this study, the diabody [CD3 x Pgp] was obtained by PCR and restriction cleavage, and expressed in E.coli 16C9. The product was purified by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry(FCM) was used to analyse the bingding properties and competitive bingding capacity. RESULTS: The sequence of diabody [CD3 x Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight(25 kD and 26 kD) in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively. CONCLUSION: The diabody [CD3 x Pgp] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesn't decrease.

[Design and activity determination of small molecular inhibitors of integrin alphavbeta3].

OBJECTIVE: To explore the design and activity determination of small molecular inhibitors of integrin alphavbeta3 through structure-based virtual screening. METHODS: Based on the crystal structure of integrin ctv33 extracellular segment in complex with an ARG-GLY-ASP ligand, docking procedure against the receptor binding domain was performed on 3D database. Integrin alphavbeta3-mediated cell adhesion assay was performed to assess the adhesion-inhibiting ability of the candidate compounds. Cell migration assay and capillary-structure-like formation inhibition assay were used to estimate the effects of the compounds on integrin alphavbeta3. Analysis of molecular graphics was carried out to deduce a probable binding model of compound with integrin alphavbeta3. RESULTS: From the top 1000 compounds with the best DOCK energy score, 50 compounds were selected for biological assay based on chemical and drug-like diversity. Seven of 50 compounds showed notable inhibition activity on cell adhesion, and two with half-maximum inhibition concentration (IC50) values less than 100 mol/L. The compound with best activity (1-37) showed high inhibitory activity in cell migration assay and capillary-structure-like formation inhibition assay. Molecular graphics analysis indicated that metal ion-dependent adhesion site (MIDAS) might be involved in the compound 1-37-mediated inhibition of ligand binding with integrin alphavbeta3. CONCLUSIONS: Through virtual screening combined with biological assay, a promising lead compound was discovered to inhibit integrin alphavbeta3, which embodies the rational drug design with computation aid and brings a new thought and approach to find novel inhibitors of integrin.

[Reversal of multidrug resistance in drug-resistant human breast cancer cell line MCF-7/ADR by calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine].

OBJECTIVE: To investigate the reversal effect of O-(4-ethoxyl-butyl)-berbamine (EBB) on multidrug resistance (MDR) in MCF-7/ADR cell. METHODS: 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the antitumor effect of EBB and determine the reversal effects of different concentrations ( < or = IC20) of EBB on MCF-7/ADR cell. Flow cytometry was applied to observe the intracellular accumulation of Rh123 and cell cycle in the presence of EBB. The expressions of MDR-related genes mdr 1 and topoisomerase II b (top II b) were evaluated by reverse transcription-polymerase chain reaction. RESULTS: The sensitivity of MCF-7/ADR to adriamycin (ADR) was enhanced up to 50. 40, 89.80, and 14.88 folds after exposure of the cells to 3 micromol/L EBB, 7.5 micromol/L EBB, and 10 micromol/L verapamil (VPL), respectively. After 2 hours of incubation with 6 micromol/L EBB, intracellular Rh123 accumulation in MCF-7/ADR cells was increased to the level comparable to that in MCF-7 cells. When 6 micromol/L EBB was added together with 2 micromol/L ADR, MCF-7/ ADR cells showed to be arrested in the G2/M phase. The declination of mdr 1 gene expression was observed when 6 micromol/L EBB, 12 micromol/L EBB, and 10 micromol/L VPL were added for 48 hours; meanwhile, the expression of top II b mRNA showed no significant change. CONCLUSION: EBB has a strong reversal effect on MDR in MCF-7/ ADR cell, which may be achieved by enhancing the arrestment of MCF-7/ADR cells at G2/M phase and increasing intracellular drug concentration.

[Cloning and expression of the extracellular domain of 4-1BBL].

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.