Construction of CAR-T cells targeting CS1 and analysis of their antitumor activity in vitro / 生物工程学报

We constructed the CS1-targeted second- and third-generation CAR-T cells with genetic engineered 4-1BB or/and ICOS as a costimulatory signaling molecule by use of lentiviral platform. The CS1-targeted second-generation CAR-T cells with ICOS or 4-1BB had similar anti-neoplastic activity. When effector/target ratio was 1:1, the CAR-T cells with ICOS showed better killing effect on IM9-lucgfp cells than those with 4-1BB. However, The CS1-targeted third-generation CAR-T cells exihibited lower cytolytic capacity against IM9-lucgfp cells than the CS1-targeted second-generation CAR-T cells when the ratio of effector/target was 1:1, 2:1 or 5:1. When the ratio of effector/target was 10:1, the killing efficacy of both the second- and third-generation CAR-T cells against IM9-lucgfp cells was more than 85%, significantly higher than that of the control T cells. Taken together, both the CS1-targeted second- and third-generation CAR-T cells with ICOS or/and 4-1BB could efficiently kill CS1-positive multiple myeloma cells, but the CS1-targeted second-generation CAR-T cells had more potent killing effect on CS1-positive multiple myeloma cells than the CS1-targeted third-generation CAR-T cells.

Study of local application of CpG-oligodeoxynucleotide combined with 4-1BB monoclonal antibody to treat hepatoma- bearing mice / 中华肝脏病杂志

Objective@#To investigate the curative effect of local application of CpG-oligodeoxynucleotide (CpG-ODN) combined with 4-1BB monoclonal antibody in hepatoma-bearing mice, and to evaluate the effect of 4-1BB monoclonal antibody on CpG-ODN immunotherapy.@*Methods@#H22 single cell suspension was injected subcutaneously into the axilla and four limbs of the BALB/c male mice to establish a tumor-bearing mice model. After 7 days, 30 mice with corresponding tumor-bearing volume were screened and randomly divided into model control group, CpG group and CpG+4-1BB group, and the drug was injected into the tumors of left lower extremity. The same batch of normal mice was selected as normal control group. Survival of mice was recorded. Tumor-bearing volume and organ index were calculated. Serum levels of interleukin (IL) - 12 and interferon (IFN) gamma and spleen CD8+T lymphocyte ratio were measured. The measurement data were analyzed by analysis of variance. The survival rate of each group of mice was analyzed by log-rank test.@*Results@#Mice in the model control group with tumor-bearing volume had a sustained growth before the execution. CpG group and the CpG+4-1BB group [(976.08 ± 29.55) mm3, (47.25 ± 0.93) mm3)] tumor-bearing volume was decreased than model group [(1 336.52 ± 39.40) mm3] (F = 5 329.273, P < 0.05). CpG+4-1BB group distant tumor-bearing volume [(611.83 ± 113.02) mm3] was decreased than model group and CpG group [(1 406.62 ± 51.09) mm3, (1 380.01 ± 51.44) mm3] (F = 247.160, P < 0.05), but there was no significant difference between the CpG group and the model group (P > 0.05). Serum IL-12 concentration (23.90 ± 2.33 pg/ml), IFN-γ concentration (103.02 ± 6.10 pg/ml) and spleen CD8+T cell ratio (4.54 ± 0.62%) in the model group were lower than those in the normal group (P < 0.05). Serum IL-12 concentration in CpG group and CpG+4-1BB group (29.21 ± 2.23 pg/ml, 37.04 ± 1.49 pg/ml), IFN-γ concentration (116.12 ± 4.08 pg/ml, 138.65 ± 1.72 pg/ml), CD8+T cell ratio (6.65 ± 0.64%, 12.73 ± 0.88%) were higher than the model group, while CpG+4-1BB group was higher than the CpG group (P < 0.05). The survival rate of CpG+4-1BB group was higher than that of model group and CpG group (χ2 = 25.544, P < 0.05), but there was no significant difference between CpG group and model group (P > 0.05). There was no significant difference in organ index between the four groups (P > 0.05).@*Conclusion@#4-1BB monoclonal antibody combined with CpG-ODN therapy can shrink hepatoma-bearing capacity, inhibit the growth of distant tumors and significantly prolong the survival time of mice.

Hu.4-1BB-Fc fusion protein inhibits allergic inflammation and airway hyperresponsiveness in a murine model of asthma / 소아과

PURPOSE: 4-1BB (CD 137) is a costimulatory molecule expressed on activated T-cells. Repression by 4-1BB is thought to attenuate Th2-mediated allergic reactions. The aim of this study was to investigate the effect of 4-1BB on allergic airway inflammation in a murine asthma model. METHODS: BALB/c mice were sensitized to and challenged with ovalbumin (OVA). Hu.4-1BB-Fc was administered 1 day before the first OVA sensitization or 1 day after the second OVA sensitization. Following antigen challenge, airway responsiveness to methacholine was assessed and bronchoalveolar lavage (BAL) fluid was analyzed. Total immunoglobulin (Ig) E, OVA-specific IgE, IgG1, and IgG2a levels in sera were measured by enzyme-linked immunosorbent assay. Lung pathology was also evaluated. RESULTS: In mice treated with Hu.4-1BB-Fc before the first OVA sensitization, there was a marked decrease in airway hyperresponsiveness, total cell count, and eosinophil count in the BAL fluid. In addition, Hu.4-1BB-Fc treatment decreased serum OVA-specific IgG1 levels and increased serum IgG2a level significantly compared with the corresponding levels in mice sensitized to and challenged with OVA. Hu.4-1BB-Fc-treated mice also showed suppressed peribronchial and perivascular inflammatory cell infiltration. In contrast, treatment with Hu.4-1BB-Fc 1 day after sensitization had no effect on airway hyperresponsiveness and showed less suppression of inflammation in lung tissue. CONCLUSION: Administration of Hu.4-1BB-Fc can attenuate airway inflammation and hyperreactivity in a mouse model of allergic airway inflammation. In addition, administration before sensitization may be more effective. These findings suggest that 4-1BB may be a useful therapeutic molecule against asthma.

The study on the lowered tumorigenicity of hepatocellular carcinoma cells cotransfected with chemokine mMIP-1α and costimulatory molecule m4-1BB L in vivo / 中华消化杂志

Objective To investigate the effects of macrophage inflammatory protein-1α (MIP-1α) combined with molecule 4-1BB L on the tumorigenicity of hepatocellular carcinoma cells in vivo. Methods Mouse MIP-1α (mMIP-1α) expressed Hepa 1-6 cells were transfected with m4-1BBL recombinant retrovirus, the anti-histidinol cells clones were selected and amplified. The expression of m4-1BB L was confirmed by flow cytometry. The growth curve of Hepa 1-6 cells transfected with mMIP-1α and m4-1BBL alone or together was drawn and compared. C57B/L Mice were randomly divided into 7 groups, 9 mice in each group, injected with mMIP-1α+m4-1BB L Hepa 1-6 cells, m4-1BB L Hepa 1-6 cells, mMIP-1α Hepa 1-6 cells, Hepa 1-6 cells, pLXSHD Hepa 1-6 cells or PBS respectively. The tumorigenicity of hepatocellular carcinoma cells and the mice survival rate were compared between each groups. Results Hepa 1-6 mMIP-1α+m4-1BB L cells which expressed both mMIP-1α and m4-1BB L were successfully established. The expression of mMIP-1α and m4-1BB L alone or together did not affect the growth curve of Hepa 1-6 cells. Observed for 5 weeks, no tumor developed in Hepa 1-6 mMIP-1α+m4-1BB L injected mice. The tumorigenicity of Hepa 1-6 mMIP-1α+m4-1BB L was lower than that of Hepa 1-6 mMIP-1α or Hepa 1-6 m4-1BB L in vivo. The survival rate of Hepa 1-6 mMIP-1α+m4-1BBL injected mice(9/9) was higher than that of Hepa 1-6 m4-1BB L injected mice (6/9)or Hepa 1-6 mMIP-1α injected mice (1/9). Conclusion Chemokine MIP-1α combined with costimulatory 4-1BB L lowered the tumorigenicity of hepatocellular carcinoma cells in vivo, and prolonged the mice survival period.

Investigation of the Effective Dose of Agonistic 4-1BB Monoclonal Antibody in a Murine Colon Cancer Metastasis Model

PURPOSE: The aim of this study was to find the dose of agonistic 4-1BB monoclonal antibody (mAb) that results in optimal T cell activation. METHODS: Cancer was induced in mice by an intrahepatic parenchymal injection of 1x10(5) cells of CT26 cells. Cancer-carrying mice (n=84) were divided into seven groups and treated with either rat IgG or agonistic 4-1BB monoclonal antibody (mAb) (5microgram, 10microgram, 20microgram, 100microgram, 200microgram, or 300microgram). All treatments were administered intraperitoneally on days 7, 9, and 11. Mice from each group were sacrificed on days 14, 28, and 42. Harvested livers were weighed and the numbers of T cells in the splenocytes were analyzed with a FACS Vantage flow cytometer. RESULTS: Liver weights increased when 5microgram of agonistic 4-1BB mAb was administered, but showed no additional weight increase for doses greater than 10microgram. The absolute numbers of CD4+ and CD8+ T cells increased in groups treated with low doses of agonistic 4-1BB mAb (5microgram, 10microgram, or 20microgram), but did not increase in the groups treated with high doses of mAb (100microgram, 200microgram, or 300microgram). The levels of CD4/annexin V and CD8/annexin V increased as the dose increased, and the absolute cell numbers of CD4/annexin V were greater than those of CD8/annexin V. CONCLUSION: Liver weight, including the cancer mass, failed to increase at agonistic 4-1BB mAb doses greater than 10microgram. A high dose (> or =100microgram) of agonistic 4-1BB mAb resulted in lower counts of absolute T cells. This study suggests that a low dose (20microgram) of agonistic 4-1BB mAb can be used for optimal T cell activation in combination with other anti-cancer treatments.

Expression of 4-1BB and 4-1BBL in thymocytes during thymus regeneration

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide-induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4+CD8+ double-positive (DP) thymocytes. Furthermore, the CD4loCD8lo, CD4+CD8lo and CD4loCD8+ thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4+CD8+ DP thymocytes present during thymus regeneration were TCR(hi) and CD69+ unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCRhi/int and CD69+/int phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4+CD8+ DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.

Location of extracellular cysteine-rich domains of 4-1BB binding to murine 4-1BB ligand and analysis of its possible structure / 中华微生物学和免疫学杂志

ObJective To locate the cysteine-rich domains(CRD) of murine 4-1BB binding to its natural ligand. Methods A serial soluble extracellular CRDs of routine 4-1BB and 4-1BBL fusion proteins was constructed and prepared. The binding of purified 4-1BB-Igs to 4-1BBL and 4-1BB monoclonal antibody were tested using ELISA assay and Western blot analysis. Blocking experiment with 4-1BBL and 4-1BB mon-oclonal antibody was performed by ELISA assay. Results All truncated overlapped proteins containing ex-tracellular CRD Ⅱ of murine 4-1BB were able to bind to 4-1BBL by ELISA assay, excepting the CRD Ⅰ do-main alone. A 4-1BB monoclonal antibody proved to block the interaction of 4-1BB and 4-1BBI, was also able to bind to CRD Ⅱ. Conclusion Murine 4-1BBL whose specificity was mapped to CRD Ⅱ of 4-1BB ex-tracellular region with a possible conformational structure.

The effect on activation of T lymphocytes with SLE by blocking 4-1BB/4-1BBL pathway / 中华微生物学和免疫学杂志

Objective To investigate the effect of costimulatory molecules 4-1BB/4-1BBL on T cell activation with systemic lupus erythematosus(SLE)patients.Methods The expression of NF-kB mRNA and p38 MAPK mRNA of T cells with 20 SLE patients and 20 normal controls before activation,after activation and blocked bv 4-1BB/4-1BBL were detected bv RT-PCR.The protein levels of p-p38 MAPK and NF-kB were detected by Western blot.Results The expression of NF-kB mRNA and p38 MAPK mRNA and the protein expression of NF-kB and p-p38 MAPK of T cell with SLE patients were higher than that of normal controls(P＜0.01).There were more expression of NF-kB mRNA,p38 MAPK mRNA,NF-kB protein and p-p38 MAPK protein of T cell with SLE patients stimulated by anti-CD3 monoantibody(P＜0.01).In anti-4-1BB monoantibody blockage group,there were decreased expression of p38 MAPK mRNA and p-p38 MAPK protein(P＜0.01).but not the decreased NF-kB mRNA and protein expression.Conclusion Costimulatory molecule 4-1BB plays a critical role in T cell activation of SLE by p38 MAPK signal pathway.

Therapeutic effect of 4-1BBmAb on immune-mediated hepatitis induced by ConA and its influ-ences on CD4+CD25+T lymphocytes / 中华风湿病学杂志

Objective To investigate the therapeutic effect of 4-1BB monoclone antibodies on mice hepatitis induced by Coneanavalin A (ConA) and its influenes on CD4+CD25+T lymphoeytes during the course. Methods The miee model of hepatic injury was indueed by ConA and monitored by hepatic function tests and hepatic pathology. The expressions of 4-1BB were examined by flow eytometry. 4-1BB monoelone antibodies were intravenously injected to the mice. The therapeutic efficacy was then examined by hepatic function tests and hepatic pathology. The expressions of CD4+CD25+T lymphoeytes were also examined by flow eytometry. Results The group of immune hepatic injury induced by ConA showed damage and marked increase of ALT and AST which were (139±22) U/L and (130±16) U/L respectively. The expression level of 4-1BB was 8.1±2.6. Compared with the eontrol group, the difference was significant (P<0.05). The overall eondition of the miee was improved after being treated with 4-1BB monoelone antibodies. ALT and AST were lowed down to (98±14) U/L and (89±11) U/L respectively and the differenee was signifieant (P<0.01). The expression of 4-1BB of the control group was 3.0±0.8 and that of the treatment group was 8.3±3.0. The difference was significant (P<0.01). Conclusion 4-1BB eontributes to the immune-mediated hepatic injury induced by Con-A.

Effect of blocking 4-1BB/4-1BBL on the proliferation of T cell and the expression of Th1 and Th2 cytokines in T cells from patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the effect of T lymphocyte proliferation,Th1 and Th2 cytokines on T cells from systemic lupus erythematosus (SLE)patients by blocking 4-1BB/4-1BBL.Methods The proliferation of T cells from 30 SLE patients and 20 normal controls was detected by MTT and the levels of patients stimulated with anti-CD3 McAb were significantly higher than those of T cells without stimulation(P＜IFN-γand IL-4 in SLE were significantly higher than those of normal controls(P＜0.01).There were more ex-pression of IFN-γ and IL-4 in supernatant of T cells from SLE after stimulated with anti-CD3 McAb(P＜0.01).However,the production of IFN-γ and IL-4 was inhibited by anti-4-1BB McAb(P＜O.05),and especially the level of IL-4 was markedly decreased.Conclusion Blocking 4-1BB/4-IBBL can significantlv inhibit the abnormal activation of T cells and the secretion of Thl and Th2 cytokines of SLE.

Expression of 4-1BB and 4-1BBL in Graves'Disease / 대한내분비학회지

BACKGROUND: 4-1BB mediated costimulatory signal is a recently identified immunotherapeutic strategy for treating autoimmune diseases without depressing the immune response. In this study, we investigated the expression of 4-1BB and 4-1BBL on the peripheral blood mononuclear cells (PBMC) and we assessed whether the serum levels of soluble (s) 4-1BB and s4-1BBL in the patients with Graves' disease (GD) and compared them with normal subjects. METHODS: Expression of 4-1BB and 4-1BBL on PBMC of GD patients was determined by flow cytometry. The concentrations of s4-1BB and s4-1BBL were assessed in the sera of GD patients by performing ELISA. RESULTS: 4-1BB was constitutively expressed on naive CD4+ and CD8+ T cells of the GD patients and this was increased by stimulation. 4-1BBL was also expressed on the antigen-presenting cells such as CD19+ B cells, monocytes and dendritic cells in GD patients. The serum levels of s4-1BB and s4-1BBL were significantly higher in GD patients than those in controls, and these levels were significantly correlated with the serum levels of thyroid-binding inhibitory immunoglobulin and free T4. CONCLUSION: These results indicate that effector T cells of GD patients can be activated through the 4-1BB-mediated costimulatory signal. Elevated s4-1BB and s4-1BBL levels in the sera of GD patients may affect modulation of the clinical course in GD patients.

CD137 induces adhesion and cytokine production in human monocytic THP-1 cells

CD137, which is expressed on activated T cells, plays a critical role in inflammatory responses. However, the exact role that CD137 plays in monocytes is not fully known. Here we studied the expression and function of CD137 in human monocytic THP-1 cells, which we found constitutively expresses CD137 at the mRNA and protein level. Cross-linking of CD137 increased the secretion of IL-8 and TNF-alpha, promoted the expression of CD54 and CD11b, and increased adhesion to extracellular matrix (ECM) proteins. In particular CD137-induced adhesion of THP-1 cells was inhibited by an inhibitor of mitogen-activated protein kinase kinase (MEK), but not by a p38 kinase inhibitor. Taken together, these results show that the adhesion and cytokine production of THP-1 cells induced by CD137 occur via activation of MEK, which results in the activation of ERK-1/2 signaling pathways. Therefore, this study suggests that CD137 induces an activating and migrating signal during inflammatory processes.

Expression of 4-1BB in the peripheral blood T lymphocytes from patients with rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the expression and role of costimulatory molecule 4-1BB on T cells of patients with rheumatoid arthritis(RA).Metheds The expression of 4-1BB on T lymphocytes from 30 RA patients and 20 healthy controls were detected by flow cytometry.Results The expression of 4-1BB on CD4~+T and CD8~+T lymphocytes from RA patients was significantly higher than that of normal control(18.56?4.08,10.33?2.13 vs 1.24?0.12,0.87?0.09,P＜0.01).There was more expression of 4-1BB on CD4~+T and CD8~+ T lymphocytes stimulated by anti-CD3 antibody from RA patients(33?4 vs 21?8,P＜0.01).In addition,the ra- tio of CD4~+T/CD8+T in RA patients was higher than that of normal controls and was positively correlated with 4-1BB~+CD4~+T cell.The expression of 4-1BB on CD4~+T in RA patients was positively correlated with the level of ESR and IgA(r=0.476,P＜0.05;r=0.659,P＜0.05).Conclusion The costimulatory molecule 4-1BB is ab- normally expressed in T lymphocytes from patients with rheumatoid arthritis.The abnormal expression of 4-1BB on T lymphocytes may play an important role in the development of RA.The expression of 4-1BB on CD4~+T cell may take part in the inflammation of RA.

Alloimmune and Skin Allograft Responses in 4-1BB (CD137)-deficient Mice

BACKGROUND: The costimulatory molecule 4-1BB, a member of nerve growth factor receptor/tumor necrosis factor (NGFR/TNFR) super family, is involved in cell survival and death. METHODS: In this study, female C57BL/6 (H-2(b)) mice were used as a recipient, and DBA/2 (H-2(d)) as a donor to assess a mixed lymphocyte reaction (MLR) and CTL response in vitro, and skin graft survival. IL-2, IFN level was measured by ELISA. RESULTS: Mixed lymphocyte reaction (MLR) analysis showed that 4-1BB- deficient responder cells showed enhanced cellular proliferation over littermate controls. In contrast, IL-2 production was diminished only in 4-1BB knockout cultures. The IFN expression, on the other hand, was comparable between the groups. When female C57BL/6 (H-2(b)) mice were grafted with the trunk skin of DBA/2 (H-2d) mice, the in vivo tissue destruction of 4-1BB-deficient mice was not distinct from the normal littermates. CONCLUSION: These data suggest that 4-1BB is critical for the induction of alloreactive responses in vitro but 4-1BB alone could not change the course of skin rejection in vivo.

Alloimmune and Skin Allograft Responses in 4-1BB (CD137)-deficient Mice

BACKGROUND: The costimulatory molecule 4-1BB, a member of nerve growth factor receptor/tumor necrosis factor (NGFR/TNFR) super family, is involved in cell survival and death. METHODS: In this study, female C57BL/6 (H-2(b)) mice were used as a recipient, and DBA/2 (H-2(d)) as a donor to assess a mixed lymphocyte reaction (MLR) and CTL response in vitro, and skin graft survival. IL-2, IFN level was measured by ELISA. RESULTS: Mixed lymphocyte reaction (MLR) analysis showed that 4-1BB- deficient responder cells showed enhanced cellular proliferation over littermate controls. In contrast, IL-2 production was diminished only in 4-1BB knockout cultures. The IFN expression, on the other hand, was comparable between the groups. When female C57BL/6 (H-2(b)) mice were grafted with the trunk skin of DBA/2 (H-2d) mice, the in vivo tissue destruction of 4-1BB-deficient mice was not distinct from the normal littermates. CONCLUSION: These data suggest that 4-1BB is critical for the induction of alloreactive responses in vitro but 4-1BB alone could not change the course of skin rejection in vivo.

The expression of 4-1BB and GITR on CD4~+CD25~(high) T cells in peripheral blood of patients with multiple sclerosis / 中国免疫学杂志

0.05).Conclusion:The decrease in 4-1BB expression,rather than the GITR expression,on CD4+CD25high T cells of MS patients suggests that 4-1BB may be involved in the lower immunoactivity of the cell population,while the GITR may play a minor or fine role in the immunoregulation of the cells.