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.
4-1BB Ligand/metabolism* ; Cell Line, Tumor ; Genetic Engineering ; Humans ; Inducible T-Cell Co-Stimulator Protein/metabolism* ; Multiple Myeloma/therapy* ; Signal Transduction ; T-Lymphocytes/chemistry* ; Xenograft Model Antitumor Assays

This study was purposed to investigate the molecular mechanism of 4-1BBL reverse signals in the human acute monocytic leukemia cell line of U937. The U937 cell line was used as target cells, and stimulated by the mouse anti-human 4-1BBL monoclonal antibody 1F1. The nuclear translocation of NF-κB and the co-location of 4-1BBL and CD28i molecules in U937 cells were observed with confocal laser scanning microscopy. The protein and m-RNA expression levels of 4-1BBL and CD28i were detected by flow cytometry and RT-PCR respectively. The results showed that the significant nuclear translocation of NF-κB and co-localization of 4-1BBL and CD28i on membrane of U937 cells appeared after being stimulated by mAb1F1. It is concluded that the 4-1BBL reverse signals transduction mediating the growth of U937 cells relates with the nuclear translocation of NF-κB. CD28i may be involved in intracellular 4-1BBL reverse signaling pathways.
4-1BB Ligand ; immunology ; metabolism ; Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; metabolism ; Coculture Techniques ; Humans ; NF-kappa B ; genetics ; Signal Transduction ; U937 Cells

BACKGROUNDBlocking the 4-1BB/4-1BB ligand (4-1BBL) signal may modulate the secretion of Th1/Th2 cytokines and prolong the survival of the grafts, which play a key role in organ transplantation tolerance. The aim of this study was to investigate the role of blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody (mAB) in acute rejection of rat orthotopic liver transplantation.

METHODSThe orthotopic liver transplantation model was set up, while male Lewis rats were used as liver donors and Brown-Norway rats as recipients. The recipient rats were intravenously injected with anti 4-1BBL mAB or isotype control antibody. Groups were monitored for graft survival after transplantation. Plasma chemistry, including aspartate transaminase (AST), alanine aminotransferase (ALT), and bilirubin (BIL), was assayed. The concentrations of interleukin (IL)-2, IL-10 and interferon (IFN)-gamma in plasma were also measured by enzyme-linked immunosorbent assay. Allograft histology images were collected under light microscope and electron microscope.

RESULTSIsotype antibody treated recipients exhibited elevated plasma levels of liver injury markers including AST, ALT and BIL, progressive portal and venous inflammation and cellular infiltration of the liver allografts, and a mean graft survival time (MST) of 10.9 days. Administration of anti 4-1BBL mAB resulted in a decrease in plasma levels of liver injury markers and the concentrations of IL-2, IL-10 and IFN-gamma. The histological grade of rejection on day 7 decreased and MST (17.3 days) increased substantially.

CONCLUSIONSThese results demonstrate that attenuation of acute rejection follows the blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody and strongly suggest it is a promising strategy to prevent progression of graft rejection by suppressing T cell-mediated immunity.

4-1BB Ligand ; immunology ; Alanine Transaminase ; metabolism ; Animals ; Antibodies, Monoclonal ; pharmacology ; therapeutic use ; Aspartate Aminotransferases ; metabolism ; Bilirubin ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Graft Rejection ; immunology ; prevention & control ; Graft Survival ; drug effects ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-2 ; blood ; Liver Transplantation ; adverse effects ; Male ; Rats ; Rats, Inbred Lew

OBJECTIVETo explore the anti-tumor immunity in vitro induced by prostate cancer cell vaccine transfected with recombinant adenovirus encoding 4-1BBL in mice.

METHODSThe replication-deficient adenovirus AdEasy-1 system was used to construct recombinant adenovirus Ad-m4-1BBL and Ad-eGFP. The prostate cancer cell RM-1 of mice was transfected with Ad-m4-1BBL and Ad-eGFP, and treated with mitomycin (MMC) to produce TCV, TCV-Ad-eGFP and TCV-Ad-m4-1BBL, followed by co-culture with syngeneic murine spleen cells. Then the cytotoxic activity of the lymphocytes against RM-1 cells was analyzed with CCK-8 solution, and IL-2 and INF-gamma were detected by ELISA.

RESULTSThe 4-1BBL protein was highly expressed in the TCV-Ad-m4-1BBL of the 4-1BBL-transfected mice. TCV-Ad-m4-1BBL significantly increased the expressions of IL-2 ([180.24 +/- 2.22] pg/ml) and INF-gamma ([1512.46 +/- 23.64] pg/ml) as compared with TCV and TCV-Ad-eGFP (P < 0.05), and induced higher RM-1 cell specific cytotoxicity ([34.24 +/- 2.64]%) than the latter two ([9.82 +/- 1.48]%) and ([14.65 +/- 3. 21]%), (P < 0.05). But none of them exhibited significant cytotoxicity against hepatocellular carcinoma Hepal-6.

CONCLUSIONThe m4-1BBL-expressing prostate cancer cell vaccine can effectively induce anti-tumor immune responses.

4-1BB Ligand ; genetics ; immunology ; Animals ; Cancer Vaccines ; genetics ; immunology ; Cell Line, Tumor ; Coculture Techniques ; Cytotoxicity, Immunologic ; genetics ; Female ; Interleukin-2 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Prostatic Neoplasms ; Transfection

OBJECTIVE: To study the expression of CD137L in laryngeal carcinoma, and to analyze its clinicopathological significance. METHOD: The expression of CD137L in 50 laryngeal carcinoma specimens and 9 normal laryngeal mucous tissues were detected by immunohistochemical staining. RESULT: The positive CD137L staining were found in all 50 cases of laryngeal carcinomas (100%), while its staining were negative in normal laryngeal mucous. There was significant difference between two groups (P < 0.01). The positive ratio of CD137L staining had no relationship with the factors such as age, sex and tumor site, while it had significant correlation with the pathological stage, T stage and lymph node metastasis. CONCLUSION The expression of CD137L might play an important role in the development of laryngeal carcinomas.
4-1BB Ligand ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis

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.
4-1BB Ligand/genetics/*metabolism ; Animals ; Antigens, CD137/genetics/*metabolism ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CD8-Positive T-Lymphocytes/cytology/metabolism ; Cell Adhesion ; Cell Differentiation ; Cell Line ; Cells, Cultured ; Cyclophosphamide/pharmacology ; Epithelial Cells/cytology ; Gene Expression Regulation ; Immunosuppressive Agents/pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; RNA, Messenger/genetics ; *Regeneration ; T-Lymphocytes/*cytology/metabolism ; Thymus Gland/*cytology/drug effects/*physiology

OBJECTIVETo study the mechanism of enhancement of the CTL activity in mice co-expressing of CD80, CD86 and CD137L genes.

METHODSThe mice were randomly divided into five groups, named A, B, C, D and E. The group A and B were control groups (CG). H22-BAL B/c HCC mouse model was established by subcutaneous injection with hepatocellular carcinoma cells of cell line H22-Wt (group A), H22-neo (group B), H22-CD80/CD86(+) (group C), H22-CD137L(+) (group D) and H22-CD80/CD86/CD137L(+) (group E), respectively. On the 14th, 35th, 56th and 84th day after the first inoculation of tumor cells, TUNEL staining and DNA ladder examination were used to detect apoptosis of splenic T lymphocytes in all groups at each post-inoculation time point. Electrophoretic mobility-shift assay (EMSA) method was used to detect the activity of nuclear factor kappaB (NF-kappaB) in splenic T lymphocytes in each group at each time point post-inoculation.

RESULTSApoptosis was found in a great number of T lymphocytes in CG on the 14th day, much more than that in group C and E. The number of apoptotic T cells in group C had a significant difference compared with that in the group E from 14th to 84th day (P = 0.003). DNA ladder analysis showed typical positive results in group C and E. The significant apoptosis fragments were found in group C on 21st, 35th and 84th days. NF-kappaB activity of T cells in groups C and E was remarkably higher than that of groups CG and D, with higher in group D than that of CG (P = 0.002), and with no significant difference between group C and E on 14th day. The activity in group E was stable and remarkably higher than that of group C on 56th and 84th days after the first inoculation.

CONCLUSIONH22-CD80/CD86/CD137L(+) induces higher NF-kappaB activity of the host T cells by synergistic action of CD28 and CD137, which may be one of the mechanisms of enhancement of the host CTL activity induced by co-expression of CD80, CD86 and CD137L genes.

4-1BB Ligand ; metabolism ; Animals ; Apoptosis ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD28 Antigens ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms, Experimental ; immunology ; metabolism ; pathology ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; metabolism ; Random Allocation ; Spleen ; pathology ; T-Lymphocytes ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; immunology

Our previous study has demonstrated that there is a significant delay of Balb/c cardiac allograft rejection in the C57BL/6 4-1BB-deficient knockout recipient. In this study, we examined the effect of combined blockade of the 4-1BB and CD28 costimulatory pathways on cardiac allograft rejection in the C57BL/6-->Balb/c model. A long-term cardiac allograft survival was induced in CD28/4-1BB- deficient mice (>100 days survival in 3 of 4 mice), which was comparable with CD28-deficient mice (>100 days survival in 2 of 5 mice; P<0.2026). There was no long-term cardiac allograft survival in either wild-type (WT) or 4-1BB-deficient mice, even though 4-1BB-deficient recipients showed a significant delay of cardiac allograft rejection than WT mice. An in vitro mixed leukocyte reaction (MLR) assay showed that 4-1BB-deficient and WT mouse T cells had a similar responsiveness to allostimulation, whereas CD28- and CD28/4-1BB-deficient mouse T cells had a defective responsiveness to allostimulation. Furthermore, 4-1BB-deficient mice showed a similar CTL but an elevated Ab response against alloantigens as compared to WT mice, and the alloimmune responses of 4-1BB-deficient mice were abrogated in the CD28-deficient background. Overall, these results indicate that the CD28 costimulatory pathway plays a major role in the alloimmune response and that 4-1BB signals are dependent upon CD28 signals.
Transplantation, Homologous/immunology ; Signal Transduction/*immunology ; Mice, Knockout ; Mice ; Isoantigens/immunology ; Heart Transplantation/immunology ; Graft Survival/immunology ; Cytotoxicity Tests, Immunologic ; Antigens, CD28/genetics/*immunology/metabolism ; Antibodies/immunology ; Animals ; 4-1BB Ligand/deficiency/genetics/*immunology/metabolism

OBJECTIVETo understand the influence of co-expression of CD80, CD86 and CD137L genes on tumor immunogenicity in hepatocellular carcinoma H22-BAL B/c mouse models.

METHODSThe mice were randomly divided into five groups, named A, B, C, D and E, and control groups A and B, 20 mice in each group. Hepatocellular carcinoma H22-BAL B/c mouse model was established by subcutaneous injection of cells H22-Wt, H22-neo, H22-CD80/CD86+, H22-CD137L+ and H22-CD80/CD86/CD137L+, respectively. The rate and incubation period of tumor development, survival rate, and the tumor growth in vivo were observed and recorded. The effects of gene transduction on immunogenicity of the tumor and antitumor immunity of the animals were assessed by re-innoculation of wild type H22 cells.

RESULTSThe rate of tumor development in group E was only 50%, much lower than that in other four groups (P < 0. 01). The tumor growth in group C was reduced with complete tumor regression in two hosts (20%, 2/10). In group E, there was more pronounced reduction of tumor size. The maximal tumor sizes were remarkably smaller than those of group C, and there was complete tumor regression in three mice (60%, 3/5). No tumor regression was found in the other three groups. Survival rates of group C and E were significantly higher than that of animals in groups A, B and D (P < 0. 01), but no significant difference was seen between group C and E. The results of re-inoculation test showed that tumor formation rate was 40% (4/10) in group C, 100% (8/8) in group D, and 0 (0/5) in group E. There were significant differences between groups C and E and control group, between group E and C, but not between C and D. After the third time of re-inoculation with H22-Wt cells at the 56th day, tumor occurred in 6/6 mice (100%) of group C, but 0 (0/5) in group E. The difference was very significant. Five animals without tumor formation after the first inoculation in group E, were re-inoculated with H22-Wt cells on the 21st day and the third re-inoculation on the 56th day, no tumor was found (0/5).

CONCLUSIONBoth co-expression and solo-expression of CD80+ CD86 and CD137L genes reduce the tumorigenicity of wild type H22 cells, but co-expression of CD80, CD86 and CD137L genes can more significantly improve the immunogenicity of H22-CD80/CD86/CD137L+ cells.

4-1BB Ligand ; genetics ; immunology ; metabolism ; Animals ; B7-1 Antigen ; genetics ; immunology ; metabolism ; B7-2 Antigen ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms, Experimental ; genetics ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Survival Analysis

OBJECTIVETo explore the possible negative regulatory elements induced by the treatment of experimental murine hepatoma with 4-1BBL, and to investigate the synergistic effects and mechanisms of 4-1BBL and soluble PD-1 (sPD-1) in tumor therapy.

METHODSMice were inoculated intramuscularly (i.m.) with 5 x 10(5) H22 tumor cells in the right hind thigh to establish the experimental hepatoma model. The mice were randomly divided into 5 groups (12 mice in each group) after inoculation. The mice of group A, B, C and D were injected with NS, plasmid pcDNA3.1, plasmid p4-1BBL and plasmid pPD-1A respectively. The mice in group E, the combinatorial treatment group, were injected with plasmid p4-1BBL and pPD-1A together. Then the anti-tumor effects, using the tumor growth rates and mice survival rates and others as parameters, were recorded. Meanwhile, the phenotype of lymphocytes and residual tumor cells in the peri-tumor tissue were analyzed.

RESULTSEither transfection with 4-1BBL gene alone or with sPD-1 alone could inhibit tumor growth to some extent, but a more significant anticancer effect was obtained in the combinatorial treatment group (group E), in which the tumors were completely inhibited in 42% of the mice, compared with 0 in the other groups. In addition, the survival rate of mice in group E was 100%, compared with 30% in group B, 65% in group C and 62% in group D. The FACS analysis results showed that the expression level of B7-H1 and B7-DC on residual tumor cells in group C (injected with p4-1BBL alone) was higher than that on cells in other groups. The amount of CD8+ T cells in the peri-tumor tissue of group E was significantly increased.

CONCLUSION4-1BBl can induce an up-regulation of negative regulatory elements and at the same time it can enhance the anti-tumor response. The combinatorial treatment with 4-1BBL and sPD-1 can produce a positive synergistic anti-tumor effect on our murine experimental hepatoma.

4-1BB Ligand ; therapeutic use ; Animals ; Antigens, Surface ; therapeutic use ; Apoptosis Regulatory Proteins ; therapeutic use ; Genetic Therapy ; Liver Neoplasms ; metabolism ; therapy ; Liver Neoplasms, Experimental ; metabolism ; therapy ; Mice ; Mice, Inbred BALB C ; Programmed Cell Death 1 Receptor