Expression of insulin-like growth factor 1 receptor in rat retina following optic nerve injury.

PURPOSE: To investigate the apoptosis in retinal ganglion cells (RGCs) and insulin-like growth factor 1 receptor (IGF-1R) in the retina following optic nerve crush. METHODS: Healthy Wistar rats (N = 70) were divided into two groups: a normal control group and an optic nerve injury group. Immunohistochemistry and flow cytometry were performed to detect the expression of IGF-1R and to measure the apoptosis of RGCs, respectively. RESULTS: Immunohistochemistry revealed that at 1 hr after optic nerve injury, IGF-1R immunoreactivity began to increase and reached a maximal level at 24 hr (p < 0.05), where it remained elevated up to 14 days after injury. RGC apoptosis in the normal control group was 0.53%, while the apoptosis rate in the optic nerve injury group increased over time. The apoptosis rate in the optic nerve injury group was 1.4% at 1 hr, 4.4% at 6 hr, 5.2% at 12 hr and reached a maximal level (8.5%) at 24 hr. Subsequently, the rate declined to 1.9% 7 days after injury and 0.9% 2 weeks after injury. CONCLUSION: The IGF-1R immunereactivity in the retina increased after optic nerve injury. IGF-1R may regulate the apoptosis and regeneration of RGCs at different stages after optic nerve injury.

Different roles of PD-L1 and FasL in immunomodulation mediated by human placenta-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from either bone marrow (BMSCs) or placenta (PMSCs) have the capacity to suppress immune responses to mitogenic and allogeneic stimulations. Both cell contact and soluble factor dependent mechanisms have been proposed to explain this immunosuppression. This study explored the roles of some of cell surface molecules expressed on human PMSCs (hPMSCs) in hPMSC mediated immunomodulation. hPMSCs strongly suppressed mitogen and allogeneic peripheral mononuclear cells (PBMCs) induced T cell activation and proliferation. hPMSCs constituently expressed programmed death-ligand 1 (PD-L1) and Fas ligand (FasL) molecules. Neutralising antibodies to-PD-L1 and FasL significantly reduced the suppressive effect of hPMSCs on T cell proliferation. However, only anti-PD-L1 antibody partially restored early T cell activation suppressed by hPMSCs. Anti-FasL antibody but not anti-PD-L1 antibody reduced apoptosis of activated T cell indicating that FasL molecule plays a role in inducing apoptosis of activated T cells, although overall hPMSCs diminished T cell apoptosis. Different effects of PD-L1 and FasL molecules on T cell activation and activated T cell apoptosis suggest that these two molecules influence T cell response at different stages. hPMSCs significantly prevented activated T cells from going into S phase. Both antibodies to PD-L1 and FasL had significant effect on reversing the effect of hPMSCs on cell cycles. hPMSCs reduced INF-γ but increased IL-10 production by mitogen activated T cells. Both antibodies partially abolished the effect of hPMSCs on INF-γ and IL-10 production. These data demonstrated that PD-L1 and FasL molecules play significant roles in immunomodulation mediated by hPMSCs. This study provides a rational basis for modulation of negative costimulators on hPMSCs to increase their immunosuppressive properties in their therapeutic applications.

[Preparation of anti-human 4-1BB monoclonal antibody and characterization of its biological activities].

AIM: To prepare an anti-human 4-1BB functional monoclonal antibody and to characterize its biological activities. METHODS: A stable human 4-1BB molecule transfected cell line 293T/4-1BB was used as an antigen to immunize BALB/c mice. By means of the cell fusion by hybridoma technique and multiple cell subcloning and repeated screening with 293T/4-1BB as the antibody screening positive cell while 293T/mock as the negative cell. The hybridoma cell lines specifically secreting anti-4-1BB monoclonal antibodies were selected. Then their characteristics and its biological activities were investigated by Western blot, fast-strip routine Ig subclass typing method, indirect immunofluorescence, competitive inhibition test, (3);H-TdR and cell apoptosis analysis. RESULTS: Three hybridoma cell lines 1G5, 4B11 and 9F11 with the property of secreting specific anti-4-1BB monoclonal antibody continuously and steadily were successfully obtained. These monoclonal antibodies could bind to human 4-1BB epitopes on activated T cells and monocytoes and DC. Additionally, mAb 4B11 could promote T proliferation and enhance the growth and maturation of Mo-DC. CONCLUSION: Three hybridoma cell lines which secrete anti-4-1BB monoclonal antibodies steadily have been established. These monoclonal antibodies could specifically recognize 4-1BB molecule and mAb 4B11 had a potent function to promote T proliferation cell as well as to enhance the growth and maturation of Mo-DC in vitro.

The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity.

Though experimental evidence shows that human bone marrow-derived mesenchymal stem cells (hBMSCs) are able to suppress T-cell activation and proliferation, the precise mechanisms are still not completely understood. Here, we investigated the role of the negative costimulatory molecule B7-H4 in the immunosuppressive effect of hBMSCs on T-cell activation. We showed that B7-H4 expresses abundantly on hBMSCs assessed by reverse transcription, immunofluorescence staining, and flow cytometric analysis. Further studies demonstrated that B7-H4 expressed on hBMSCs inhibits T-cell activation and proliferation via induction of cell cycle arrest and inhibition of NF-kappaB nuclear translocation. Blocking B7-H4 would decrease the secretion of transforming growth factor-beta1 (TGF-beta1) in the supernatant of activated T cells co-cultured with hBMSCs. Addition of neutralizing antibodies against B7-H4 significantly attenuated the inhibitory effects of hBMSCs on T-cells. Thus, our study established the novel role of B7-H4 molecule in the suppressive effect of hBMSCs on T-cell activation and proliferation. Taken together, these results highlight the complex role of hBMSCs in regulating the immune response, asserting the possibility of their therapeutic application in transplantation, the treatment of graft-versus-host disease (GVHD), and autoimmune diseases.

Fine-tuned expression of programmed death 1 ligands in mature dendritic cells stimulated by CD40 ligand is critical for the induction of an efficient tumor specific immune response.

During maturation, murine myeloid dendritic cells (DCs) upregulated the expressions of CD11c, CD25, CD40, CD80, CD86, MHC II and programmed death 1 ligands 1 and 2 (PD-L1 and PD-L2). Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-alpha. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-alpha stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-ligated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation.

[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.

A novel anti-human syndecan-1 (CD138) monoclonal antibody 4B3: characterization and application.

Syndecan-1 (CD138), a member of integral membrane heparin sulfate proteoglycans, is an essential matrix receptor for maintaining the normal morphological phenotypes. In this study, we generated a specific mouse anti-human syndecan-1 monoclonal antibody (mAb) 4B3 and identified it by competition assay with the available syndecan-1 mAb (BB4). Stained by 4B3, the expression of syndecan-1 was detected on tumor cell lines, such as 8226, U266, XG-1, XG-2, Daudi and Jurkat. The expression was also found on neuron stem cells. It was established that 4B3 mAb could inhibit XG-1 and XG-2 proliferation. The data not only determined that 4B3 mAb was a functional anti-human syndecan-1 mAb, but also indicated that syndecan-1 might be a valuable surface antigen and play an important role in regulation of tumor pathology and differentiation of neural stem cells. This novel antibody 4B3 may be value of study of tumor proliferation/survival mechanism and contributes to diagnosis and treatment of diverse diseases.

[Effects of MSC on cell cycle, phenotype and cytokine secretion of T cells activated by PHA].

AIM: To study the effect of human bone marrow derived mesenchymal stem cell (MSC) on T cell cycle and activation, and to investigate the inhibitory effect of MSC on T cell proliferation and the underlying mechanism. METHODS: Human bone marrow derived MSC were isolated by gradient centrifugation. then in vitro MSC were cultured, expanded,and were used in test after third passage. FCM analysis and ELISA were used to investigate the effects of MSC on the early activation marker expression of T cells, cell cycle and cytokine secretion. RESULTS: T cells stimulated by PHA in the presence of MSC were arrested at G0/G1 phase. The expression of the early activation marker CD25 and CD69 of T cells was inhibited in the presence of MSC both in CD4(+) and CD8(+) T cell subpopulation. MSC caused a sharp decrease of cytokine secretion in IL-2 and IFN-gamma. CONCLUSION: Human bone marrow derived MSC can suppress the activation and proliferation of T cells by altering T cell cycle.

[Construction of the transfected cell line expressing the human CXCR4 gene and study of its biological function].

AIM: To construct the tranfected cell line expressing the human CXCR4 gene and to study the biological function. METHODS: The total RNA was isolated from peripheral blood mononuclear cell (PBMC) with TRIzol, and the CXCR4 gene was amplified by RT-PCR, then digested with restriction endonuclease Pst I and EcoR I, and inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was co-transfected into the package cells 293T with LipofectAMINE 2000. Then the supernatant of the 293T cell culture was used to infect L929 cells, the cell clones stably expressing the CXCR4 molecule were screened in the presence of Zeocin (500 mg/L) after 72 h cultivation. RESULTS: It was found that the full-length of CXCR4 gene was successfully cloned, and the recombinant retrovirus vector carrying the CXCR4 gene was constructed. The CXCR4 cDNA transfected L929 cell could stably express the human CXCR4 on the cell membrane, and the migration ability of transfected cells was well evidenced in the transwell system induced by SDF-1alpha after the transfection with CXCR4. CONCLUSION: The CXCR4 transfected L929 cell line was successfully established, and it can make the basis for the further research.

[Biological effects of soluble CD40 ligand on lung cancer cell line A549 and its mechanism].

BACKGROUND & OBJECTIVE: Although the roles of CD40 in B cells have been intensively studied, little is known on the function of CD40 in lung cancer cell lines. This study was to investigate biological effects of soluble CD40 ligand (sCD44L) on lung cancer cell line A549 (CD40 positive), and its possible mechanism. METHODS: A549 cells were co-incubated with sCD40L, cell proliferation was detected by MTT assay and 3H-TdR incorporation method. Immunofluorescence technique and flow cytometry (FCM) were used to evaluate changes in cell phenotypes and cell cycle. Cell apoptosis, and expression changes of Bcl-2 and Bax were observed by FCM, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blot. RESULTS: Compared with control cells, proliferation of A549 cells co-incubated with sCD40L was inhibited (P< 0.05). Positive rates of cell surface molecules, CD49e, CD54, TNFRI, and CD95L, in A549 cells co-incubated with sCD40L for 72 h were (61.2+/-4.8)%, (31.2+/-6.1)%,(42.7+/-5.9)%, and (38.2+/-3.4)%, respectively, while those in control cells were (34.7+/-2.1)%, (7.1+/-1.6)%, (15.2+/-4.1)%, and (10.1+/-2.3)%, respectively (P< 0.05). However, positive rate of TNFRII in A549 cells co-incubated with sCD40L[(8.7+/-0.8)%] was lower than that in control cells [(58.1+/-3.6)%] (P< 0.05). G1 phase of A549 cells treated with sCD40L for 72 h was (76.0+/-9.1)%, more than that of control cells [(56.7+/-6.9)%], while S phase of sCD40L-treated A549 cells [(10.3+/-5.7)%] was less than that of control cells [(32.7+/-5.5)%]. No significant apoptosis of A549 cells was observed after co-incubated with sCD40L for 72 h, but Bax expression was up-regulated. CONCLUSION: sCD40L may inhibit cell proliferation, cause changes in phenotype and cell cycle of A549 cells, and alter expression of apoptosis-associated gene, such as Bax.

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.

[Expression of CXCR4 and its effect on the biological behavior of multiple myeloma cells].

OBJECTIVE: To investigate the effect of stromal cell derived factor-1alpha(SDF-1alpha) expression and its receptor CXCR4 on the biological behavior of multiple myeloma (MM) cells and on the expression of soluble intercellular adhesion molecule 1 (ICAM-1). METHODS: FACS analysis was used to study the expression of ICAM-1 (CD(54)) and CXCR4 on the surface of MM cells. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the soluble ICAM-1 level. RESULTS: (1) Fresh MM cells expressed variable levels of functional CXCR4 [(50.4 +/- 27.3)%], which was correlated with the in vitro ability of transwell migration of MM cells [(23.6 +/- 17.2)%, P < 0.01]. (2) SDF-1alpha could up-regulate the expression of ICAM-1 on MM cells. Furthermore, the serum level of sICAM-1 was correlated with the expression of CXCR4 on MM cells. CONCLUSION: SDF-1alpha/CXCR4 plays an important role on the biological behavior of MM cells via mediating the effect of adhesion molecules.

[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.

Melanocytes are potential immunocompetent cells: evidence from recognition of immunological characteristics of cultured human melanocytes.

The dendritic nature, the strategic location, and other accumulated evidence about the immunologic characteristics of melanocytes suggest that they are not only professional melanin producing cells but are also immunocompetent cells. In this study, we demonstrated that cultured melanocytes express low levels of some immunologically important surface markers such as intercellular adhesion molecule-1 and CD 40. Moreover, we report for the first time CD 40 expression by melanocytes can be up-regulated by interferon-gamma (IFN-gamma) stimulation. Optimal enhancement of CD 40 expression was observed at an IFN-gamma concentration of 300 U/ml after a co-culture period of 72 h. Maximal melanocyte-driven T lymphocyte proliferation and interleukin-12 secretion were also observed following the same treatment and proved to be CD 40-dependent. Our data further suggest that upon CD 40 ligation, melanocytes up-regulate their co-stimulating and adhesion molecules. In addition to previous descriptions about the melanocyte's antigen processing and presenting capacity, we therefore hypothesize a dynamic model in which melanocytes alternatively work as heterogeneous antigen presenting cells. As a result of CD 40 expression on the cell surface, melanocytes might contact and subsequently stimulate CD8+ cytotoxic T lymphocytes directly via CD 40-CD 40 L interaction in some cases.