Immunogenicity moderation effect of interleukin-24 on myelogenous leukemia cells.

Previous studies have shown that interleukin-24 (IL-24) has tumor-suppressing activity by multiple pathways. However, the immunogenicity moderation effect of IL-24 on malignant cells has not been explored extensively. In this study, we investigated the role of IL-24 in immunogenicity modulation of the myelogenous leukemia cells. Data show that myelogenous leukemia cells express low levels of immunogenicity molecules. Treatment with IL-24 could enhance leukemia cell immunogenicity, predominantly regulate leukemia cells to produce immune-associated cytokines, and improve the cytotoxic sensitivity of these cells to immune effector cells. IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. Moreover, IL-24 had marked effects on downregulating the expression of angiogenesis-related proteins vascular endothelial growth factor, cluster of differentiation (CD) 31, CD34, collagen IV and metastasis-related factors CD147, membrane type-1 matrix metalloproteinase (MMP), and MMP-2 and MMP-9 in transplanted tumors. These findings indicated novel functions of this antitumor gene and characterized IL-24 as a promising agent for further clinical trial for hematologic malignancy immunotherapy.

Antitumor effect and underlying mechanism of RGD-modified adenovirus mediated IL-24 expression on myeloid leukemia cells.

Interleukin-24 (IL-24), a member of the IL-10 cytokine gene family, causes growth suppression and apoptosis in various solid tumor cells. However, the effects of IL-24 on hematopoietic malignant cells have not been extensively explored. In this report, we constructed an RGD-engineered recombinant adenoviral vector, Ad.RGD-IL-24, and assessed its effects on human myeloid leukemia cells. Ad vector mediates gene transfer into leukemia cells with high efficiency. Ectopic over-expression of IL-24 has significant growth inhibition and differentiation inducement effects on these cells. Treatment with Ad.RGD-IL-24 could potentially induce leukemia cells' cell-cycle arrest. In addition, IL-24 expression could significantly induce apoptosis of the THP-1 cells. Ad.RGD-IL-24 had a potent effect on the up-regulation of the expression of GRP78/Bip, GADD34 and Bax, down-regulation of the expression of Bcl-2 and Mcl-1, and induced the activation of Caspase-3, which may be responsible for its apoptosis-inducing effect on THP-1 cells. Furthermore, IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. These findings showed the marked antitumor activity of IL-24 and provided potential perspectives in designing therapeutic or vaccine strategies in immuno-gene therapy of myeloid leukemia.

Preparation and characterization of monoclonal antibody against human B7-H4 molecule.

B7-H4, a member of B7 family, is widely expressed in tumor tissues and plays an important role in the negative regulation of T cell immunity. In this study, we report on the establishment and characterization of a functional anti-human B7-H4 monoclonal antibody (MAb) 5G3 through hybridoma method. Flow cytometry analysis showed that MAb 5G3 specifically bound to B7-H4 molecule. Functional experiments indicated that MAb 5G3 could block the inhibitory role of B7-H4 molecule on A549 cells in and reduce the apoptosis of Jurkat cells, suggesting that MAb 5G3 is an antagonistic antibody and a useful tool for further studies of B7-H4 functions in cancers.

Preferential expansion of umbilical cord blood-derived CD34-positive cells on human leukemia inhibitory factor transgenic feeder cells cultured on regenerated silk fibroin film.

In vitro expansion of transplantable hematopoietic stem cells (HSCs) is a very promising approach for different clinical applications. We have recently developed a new culture system that facilitates in vitro expansion of transplantable cord blood HSCs. In our study, we constructed a recombinant adenovirus Ad-GFP/human leukemia inhibitory factor (hLIF) expressing hLIF. The hLIF gene was delivered into human embryo lung fibroblast cell line WI-38 via infection with Ad-GFP/hLIF. Then, the transgenic cells were cultured on regenerated silk fibroin (SF) films as feeder layer cells for expansion of cord blood CD34(+) cells. Our results showed that the hLIF transgenic WI-38 cells cultured on SF could express hematopoiesis-related cytokines at higher levels compared with control groups. The hLIF-expressing feeder layer cells cultured on SF in combination with cytokines more efficiently expanded CD34(+) cells and CD34(+) CD38(-) cells. The percentages of adhesion molecules on the expanded CD34(+) cells in transgenic feeder layer cells cultured on SF were higher than those of control groups. Interestingly, the migration rate assessed by transwell assay was also significantly higher than those of control groups, which suggests that transgenic feeder layer cells cultured on SF has powerful ability to maintain the homing capacity of expanded CD34(+) cells.

[Preparation and characterization of anti-human B7-H4 monoclonal antibodies].

AIM: To prepare anti-B7-H4 monoclonal antibodies (mAbs) and to characterize their biological functions. METHODS: A human B7-H4 transfectant cell line L929/B7-H4 was used as an immunogen to immunize BALB/c mice. By means of the B lymphoma hybridoma technique, immunofluorescent cytometry, repeated screening and multiple subcloning, the hybridoma cell lines specifically secreting anti-B7-H4 mAbs were screened. The fast-strip analysis was used to investigate murine Ig subclass. The specificity of mAbs was determined by Dot-blot and Western blot. Competitive inhibition test was employed to identify mAb binding sites on B7-H4. T cell proliferation inhibition blocking test was used to examine mAb biological function. RESULTS: Two hybridoma cell lines were obtained and named 1F10 and 2B2, respectively. They could secret continuously and stably specific anti-B7-H4 mAbs. The result of Dot-blot indicated that two mAbs could recognize specifically B7-H4, but only mAb 2B2 recognized B7-H4 when using Western blot. The competitive inhibition test showed that two mAbs bound different epitopes on B7-H4. The two mAbs could partially block the inhibitory effects of B7-H4 on T cell proliferation in vitro. CONCLUSION: Two hybridoma cell lines secreting anti-B7-H4 mAbs were obtained. Obtained two mAbs provided useful tool for further studying B7-H4's biological functions.

[Preparation of three functional monoclonal antibodies against human FL molecule and analysis of their bioactivity].

AIM: To Prepare three functional monoclonal antibodies(mAbs) against human FL molecule and analyze their bioactivity. METHODS: The cell line L929-FL transfected with human FL gene was used as immunogen. The hybridomas secreting the antibodies against human FL were obtained by fusing splenoecytes from the immunized mice with murine myeloma cells(Sp2/0). Their subclasses were analyzed using fast-strip method. The monoclonal antibodies were produced in mouse peritoneal cavity and purified by Protein G affinity chromatography. The inhibitory effect of mAbs against FL on leukemia cell lines U937 and HL-60 was detected by MTT. The apoptosis of U937 and HL-60 cells stained by annexin-V/PI was determined by FCM. RESULTS: Three hybridomas named 3C2, 3C6 and 8D10 were successfully obtained, which secreted monoclonal antibodies against human FL molecule stably. Their subclasses were the mouse IgG2a with kappa light chains. The three monoclonal antibodies recognized the FL molecule on U937 and HL-60 cells that also coexpressed Flt3 molecule. When U937 and HL-60 cells were cultured in presence of 3C2, 3C6 and 8D10, their proliferation was reduced as compared to that in control in MTT assay(P < 0.05). The analysis of annexin-V/PI binding to U937 and HL-60 cells by FCM showed the mAbs had the apoptotogenic activity of the monoclonal antibodies against human FL molecule. CONCLUSION: 3C2, 3C6 and 8D10 are three funtional monoclonal antibodies against human FL molecule. They may be of some value in the study of the roles of FL/Flt3 interaction in leukemia pathogenesis.

[Therapeutic effect of agonistic CD40 monoclonal antibody combined with CTL on hu-SCID mouse B lymphoma model].

OBJECTIVE: To study the therapeutic effect of agonistic CD40 monoclonal antibody combined with tumor specific cytotoxic T lymphocyte (CTL) on B lymphoma. METHODS: Human B lymphoma cell line, Daudi cells, were cultured with CD40 mAb (5C11) for 24 and 48 hours, respectively. Annexin V/PI-binding assay was employed to analyze apoptosis, and FCM to analyze Fas (CD95) expression. Human peripheral monocyte-derived DC were loaded with apoptotic Daudi cells and stimulated by SC11 for further maturation. Tumor specific CTL were generated in vitro by co-culture of mature DC with autologous T lymphocytes. DNA fragmentations of Daudi cells treated with 5C11, CTL or 5C11 combined with CTL were determined by JAM assay. To establish the B lymphoma model, Daudi cells were subcutaneously injected into humanized SCID mice (hu-SCID). 1 or 3 weeks after tumor transfer. tumor-bearing mice were respectively treated with SC11, CTL, 5C11 combined with CTL by intraperitoneal injection. Tumor volume in differently treated mice was measured every week after therapy, and the survival of tumor-bearing mice was recorded. RESULTS: 5C11 significantly up-regulated FAS expression in Daudi cells, but had no significant effect on apoptosis rate of Daudi cells. Tumor-specific CTL could effectively kill Daudi cells. Fragmentation of Daudi cells co-cultured with CTL was remarkably enhanced by combination with SC11. Tumor growth in hu-SCID mice was apparently delayed by treatment with SC11, CTL, or SC11 combined with CTL. Moreover, minimal tumor burden mice got 30.0% or 70.0% complete remission (CR), respectively, when received CTL treatment or combination treatment of SC11 with CTL, and the lifespan of tumor bearing mice was also prolonged significantly. CONCLUSION: SC11 may enhance the sensitivity of Daudi cells to apoptosis by up-regulation of Fas expression and promote cytotoxicity of CTL in vitro and therapeutic effect in vivo.

A functional anti-human 4-1BB ligand monoclonal antibody that enhances proliferation of monocytes by reverse signaling of 4-1BBL.

4-1BB Ligand (4-1BBL), a transmembrane molecule, member of the tumor necrosis factor ligand superfamily, is an important costimulatory molecule in the immune response. In this study a functional anti-human 4-1BBL MAb 1F1 was obtained and the specificity of this MAb was verified by flow cytometry and Western blotting. This MAb effectively recognized the 4-1BBL molecule expressed on a series of malignant cell lines as well as on DC and monocytes and it inhibited the proliferation of T lymphocytes, costimulated by soluble 4-1BBL and agonist anti-human CD3 MAb. Furthermore, we demonstrated that MAb 1F1 induced an impressive proliferation of monocytes from peripheral blood by triggering the reverse signal through 4-1BBL. This functional anti-human 4-1BBL MAb provides a valuable tool for further study of biological functions as well as signal transduction of 4-1BBL/4-1BB.

[Effect of agonist anti-CD40 mAb 5C11 on the induction and biological characteristics of leukemic dendritic cells].

OBJECTIVE: To study the impact of an agonist anti-CD(40) monoclonal antibody 5C11 on the induction and biological characteristics of leukemic dendritic cells. METHODS: Combinations of 5C11 and different cytokines were used to induce differentiation of leukemic blasts into dendritic cells. Morphology was observed by light microscopy. Surface antigens of the induced cells were analyzed by fluorescence-activated cell sorting (FACS), the yields of dendritic cell by cell counting, the levels of IL-6 and IL-12 by ELISA, T cell proliferating activity by allo-mixed lymphocyte reaction (MLR) in vitro. Allogeneic T cells were stimulated with leukemic dendritic cells and T-cell cytotoxicity was measured by MTT assay. RESULTS: When cultured with combinations of 5C11 and different cytokines, the leukemic cells isolated from the patients could differentiate into dendritic cells. The morphology showed typical features of dendritic cells, which expressed high levels of CD(40), CD(80) and CD(86). In comparison with the original leukemia cells, the leukemic dendritic cells secreted less IL-6 but more IL-12 (P < 0.05). The leukemic dendritic cells were potent to stimulate the proliferation of allogeneic T cells, and the latter was able to lyse the original leukemia cells. CONCLUSION: Leukemic blasts could be induced to differentiate into functional dendritic cells. It may be of great value in the adoptive immunologic therapy of leukemia.

gp130-linked signal transduction promotes the differentiation and maturation of dendritic cells.

In order to explore the role of gp130-linked signal transduction in the differentiation and maturation of dendritic cells (DC), the mAb, B-S12, an agonist of gp130, was used for the activation of gp130 on DC. The effects of cytokines and of anti-gp130 mAb on the proliferation of DC, and their expression of IL-12 and CD80 (B7-1) by DC were evaluated. DC differentiating from peripheral blood mononuclear cells did not express the IL-6 receptor alpha chain, but expressed gp130. Anti-gp130 mAb promoted the proliferation of DC, induced by IL-4 and granulocyte macrophage colony stimulating factor (GM-CSF), by up-regulating the GM-CSF receptor on DC. DC induced by gp130 mAb and cytokines expressed DC-derived CC chemokine, as measured by RT-PCR. Induced DC also stimulated strong proliferation of autologous T cells in mixed lymphocyte reaction since an up-regulated expression of IL-12 and CD80 (B7-1) was observed in DC activated by anti-gp130 mAb. Thus, gp130 signal transduction is important for the differentiation and maturation of DC.