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

Now, it has been being accepted that reverse signaling through CD137 ligand (CD137L) plays an important role in vivo during hematopoiesis and in immune regulation. However, due to technical difficulty in dissecting both directional signaling events simultaneously in vivo, most biological activities caused by CD137-CD137L interactions are considered as results from signaling events of the CD137 receptor. To make the story more complex, CD137-/- and CD137L-/- mice have increased or decreased immune responses in a context-dependent manner. In this Mini review, I will try to provide a plausible explanation for how CD137L signaling is controlled during immune responses.
4-1BB Ligand* ; Animals ; Hematopoiesis ; Inflammation ; Mice

OBJECTIVEThe non-specific antitumor immunity effect of 4-1BBL-B7-H3 gene was investigated by establishing an oral squamous cell carcinoma human peripheral blood lymphocyte-severe combined immunodeficient (SCID) mice chimeric model.

METHODSForty mice were randomly divided into five groups. All groups, except the non-immune reconstitution group (group D), had reconstructed human partial immune system. The control group (group A) was injected with Tca8113 cells. The Ad4-1BBL-B7-H3 group (group B) was injected with Tca8113 cells transfected by adenovirus containing 4-1BBL-B7-H3 gene. The empty vector group (group C) was injected with Tca8113 cells transfected by adenovirus containing an empty vector. The non-immune reconstitution group (group D) was injected with Tca8113 cells. The non-tumor group (group E) was injected with PBS. The tumor volumes in each group were measured weekly. Human IgG in blood was obtained through the tail vein and was determined by enzyme-linked immunosorbent assay. Human CD3+ and D56 lymphocytes were assessed by flow cytometry. Model animals were killed on the ninth week. Differences in the expression of the natural killer group 2 member D (NKG2D) and toll-like receptor 2 (TLR2) in tumor tissues of each group were observed by immunohistochemical method. 4-1BBL-B7-H3 gene expression in mice tumor tissues was detected by reverse transcription polymerase chain reaction (PCR) and the expressions of major histocompatibility complex 1 class related molecule (M1C) A, M1CB, and TLR2 were detected by real-time quantitative PCR.

RESULTSThe tumor volumes of group B were remarkably lower than those in the other groups (P < 0.05). Human IgG and CD3+ and CD56+ lymphocytes were detected in the peripheral blood of immune-reconstituted mice. These lymphocytes were remarkably higher in group B than those in groups A, C, and E (P < 0.05). Higher NKG2D and TLR2 expression were observed in group B tumor than those in the other groups. The stable expression of 4-1BBL-B7-H3 gene in group B was proven. The expression of M1CA, M1CB, and TLR2 were significantly higher in the group B tumor than those in groups A, C, and D (P < 0.05).

CONCLUSIONThe high 4-1BBL-B7-H3 gene expression in tumor tissues could successfully induce the proliferation of CD3+ and CD56+ lymphocytes. This expression can also directly or indirectly activate TLR2 and up-regulate the expression of NKG2D and its ligands (M1CA and M1CB), which result in an effective antitumor immune response.

4-1BB Ligand ; Adenoviridae ; Animals ; Carcinoma, Squamous Cell ; Disease Models, Animal ; Genetic Vectors ; Humans ; Mice ; Mice, SCID ; Mouth Neoplasms ; Neoplasms ; Transfection

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

BACKGROUND: Natural killer cells expanded from human peripheral blood (PB) have been used in cancer immunotherapy research. Although most research teams have access to human PB, it is necessary to find a source of blood that can be easily obtained. We have tested the possibility of using blood retained in a disposable platelet apheresis set as an alternative source, with special interest in expansion of NK cells for use in cancer immunotherapy research. METHODS: For expansion of NK cells, peripheral blood mononuclear cells (PBMCs) were isolated from an MCS+ platelet apheresis kit (Haemonetics, Braintree, USA) and PB from the same donor (n=7) and co-cultured with 100-Gy gamma ray-irradiated K562 cells expressing the 4-1BB ligand and membrane-bound IL-15 for three weeks in RPMI1640 medium in the presence of IL-2 and IL-15. Cytotoxicity was measured using WST-1 at 1:1, 2:1, and 4:1 effector-to-target (E:T) ratios for a period of four hours. RESULTS: Mean rate of expansion of NK cells was 1,097-fold and their purity was 94.4% from blood retained in a disposable platelet apheresis set; mean rate of expansion of NK cells was 953-fold and their purity was 92.0% from PB after a period of three weeks. No differences in cytotoxicity against K562, 697, Raji, and RPMI8226 were observed between NK cells expanded from two blood sources. CONCLUSION: Blood retained in a disposable platelet apheresis set is a useful and convenient source for expansion of NK cells for use in cancer immunotherapy research.
4-1BB Ligand ; Blood Component Removal ; Blood Platelets ; Humans ; Immunotherapy ; Interleukin-15 ; Interleukin-2 ; K562 Cells ; Killer Cells, Natural ; Tissue Donors

BACKGROUND: The ligand for CD137 (CD137L; also called 4-1BBL) is mainly expressed on activated APCs such as dendritic cells, B cells and macrophages. Even though CD137L functions as a trigger of the CD137 signaling pathway for T cell activation and expansion, engagement of CD137L can deliver a signal leading to the production of proinflammatory cytokines in macrophages. METHODS: We generated cell-permeable TAT-CD137L cytoplasmic domain fusion protein (TAT-CD137Lct) and examined its ability to initiate the CD137L reverse signaling pathway. RESULTS: Treatment of TAT-CD137Lct induced the production of high levels of IL-6 and TNF-alpha mRNAs and proteins in peritoneal macrophages. TAT-CD137Lct increased phosphorylation of Erk, p38 MAPK and Jnk, and activated transcription factors C/EBP and CREB. However, TAT-CD137Lct did not visibly affect the degradation of the inhibitor of NF-kB (IkBalpha). We further demonstrated that JNK activation was required for TAT-CD137Lct-induced production of TNF-alpha, while activation of Erk and p38 MAPK were involved in IL-6 and TNF-alpha production. CONCLUSION: Our results suggest that TAT-CD137Lct is an effective activator for the CD137L reverse signaling pathway.
4-1BB Ligand ; B-Lymphocytes ; Cytokines ; Cytoplasm ; Dendritic Cells ; Interleukin-6 ; Macrophages ; Macrophages, Peritoneal ; NF-kappa B ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Proteins ; RNA, Messenger ; Transcription Factors ; Tumor Necrosis Factor-alpha

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 investigate the adjuvant effect of co-stimulatory molecule CD137L on cellular responses to HBsAg DNA vaccination in mice.

METHODSEukaryotic expression vector containing the full length of mouse CD137L cDNA sequence (pcD137L) was transfected into NIH3T3 cells, and then the expression of CD137L mRNA and protein in the transfected cells were detected by RT-PCR, flow cytometry and immunofluorescence method, respectively. The BALB/c mice were co-immunized with pcD137L and HBsAg DNA vaccine (pcDS) by intramuscular injection. HBsAg-specific activity of splenic cytotoxic T lymphocyte (CTL) in the immunized mice was measured by LDH release assay. The splenic memory CD8+ T cells, and intracellular IFN-gamma and IL-4 of splenic lymphocytes and CD8+ T cells after immunization were detected by flow cytometry.

RESULTSThe NIH3T3 cells transfected with pcD137L efficiently expressed mouse CD137L mRNA and protein. HBsAg-specific CTL responses induced by the pcDS plus pcD137L group were much stronger than those induced by pcDS alone at a week after immunization (P<0.05). Compared to mice immunized with pcDS alone, CD44high and CD127(IL-7R) were all significantly up-regulated in memory CD8+ T cells from the mice immunized with pcDS combined CD137L both at a week and 12 weeks after immunization (P<0.05 and P<0.01). The pcDS plus CD137L group also elicited higher levels of IFN-gamma secreted by CD8+ T cells and splenic lymphocytes than pcDS alone at a week, 12 and 13 weeks after immunization, respectively (all P<0.01).

CONCLUSIONDNA, viral/immunol; Co-stimulatory molecule CD137L can enhance the Tc1 (type I) cell-mediated immunity, HBsAg-specific CTL and memory responses induced by HBsAg DNA vaccine, and may be an efficient adjuvant in priming HBV-specific T cell response.

4-1BB Ligand ; immunology ; pharmacology ; Adjuvants, Immunologic ; pharmacology ; Animals ; Female ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccination ; methods ; Vaccines, DNA ; immunology

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